Physical activity at diagnosis is associated with tumor downstaging after neoadjuvant chemoradiotherapy in patients with rectal cancer.

BACKGROUND: Patients with rectal cancer are often treated with neoadjuvant chemoradiotherapy, followed by a waiting period and surgical resection. Good or complete response to neoadjuvant chemoradiotherapy might enable organ preservation, which highlights the need to increase response rates. Pre-clinical studies suggest that physical activity during neoadjuvant chemoradiotherapy may improve tumor downstaging. PURPOSE: To investigate whether physical activity and physical functioning of patients with rectal cancer at diagnosis are associated with tumor downstaging after neoadjuvant chemoradiotherapy. MATERIALS AND METHODS: Patients were included if they participated in the Dutch Prospective ColoRectal Cancer Cohort, a nationwide cohort providing an infrastructure for scientific research, and received neoadjuvant chemoradiotherapy for rectal cancer. Tumor downstaging was dichotomized into good/complete or moderate/poor downstaging. Physical activity (total physical activity, moderate-to-vigorous physical activity (MVPA), and Dutch physical activity guideline adherence) and physical functioning were assessed using questionnaires. Logistic regression analyses were performed to examine associations of physical activity and physical functioning with tumor downstaging, adjusted for relevant confounders. RESULTS: 268 patients (aged 62 ± 11 years, 33 % female) with rectal cancer were included. Patients with moderate (OR = 2.07; 95%CI = 1.07 - 4.07; p = 0.03) or high (OR = 2.05; 95%CI = 1.05 - 4.07; p = 0.04) levels of MVPA were more likely to have good/complete tumor downstaging than patients with low levels. No significant associations with tumor downstaging were found for total physical activity, Dutch physical activity guideline adherence, and physical functioning. CONCLUSIONS: We found augmented tumor downstaging in patients with rectal cancer with moderate or high levels of self-reported MVPA before the start of neoadjuvant chemoradiotherapy compared to patients with low levels.

Automated, Point-of-Care mobile flow cytometry: Bringing the laboratory to the sample.

Background: Innate effector cells are very responsive to infectious and inflammatory cues found in damaged and inflamed tissues. Their activation is a potential target to assess the state of the immune system. Unfortunately, these cells are very susceptible for ex-vivo activation, hampering accurate interpretation of flow cytometry data. Whether a brief window exists before ex-vivo activation starts to occur is currently unknown. Aims: 1) This study extensively investigated ex-vivo activation of innate effector cells over time. 2) We tested the feasibility of applying a mobile, automated, flow cytometry laboratory for out-of-hospital Point-of-Care analyses to minimize ex-vivo activation bias. Methods: 1) Ex-vivo neutrophil, eosinophil and monocyte activation in a blood collection tube over time and the reactivity to a formyl-peptide was investigated in a healthy cohort. 2) To facilitate fast, out-of-hospital analysis, application of the mobile flow cytometry was tested by placing an automated flow cytometer into a van. The stability of the setup was assessed by repetitively measuring laser alignment and fluorescence verification beads. Findings: 1) Immediately after venipuncture activation marker expression on neutrophils, eosinophils and monocyte subsets started to change in a time-dependent manner. 2) The mobile flow cytometry laboratory travelled over 3000 km, performing measurements at 19 locations with a median single-person-set-up time of 14 min. The laser alignment and fluorescence were stable during all experiments. Conclusions: Accurate flow data of innate immune cells are only obtained when ex-vivo activation is kept to minimum. The use of a mobile, fast, automated, flow cytometry laboratory for out-of-hospital Point-of-Care analyses provides new investigational and diagnostic possibilities outside major hospital flow cytometry laboratories.

Neutrophil and Eosinophil Responses Remain Abnormal for Several Months in Primary Care Patients With COVID-19 Disease.

Introduction: Neutrophil and eosinophil activation and its relation to disease severity has been understudied in primary care patients with COVID-19. In this study, we investigated whether the neutrophil and eosinophil compartment were affected in primary care patients with COVID-19. Methods: COVID-19 patients, aged ≥ 40 years with cardiovascular comorbidity presenting to the general practitioner with substantial symptoms, partaking in the COVIDSat@Home study between January and April 2021, were included. Blood was drawn during and 3 to 6 months after active COVID-19 disease and analyzed by automated flow cytometry, before and after stimulation with a formyl-peptide (fNLF). Mature neutrophil and eosinophil markers at both time points were compared to healthy controls. A questionnaire was conducted on disease symptoms during and 3 to 6 months after COVID-19 disease. Results: The blood of 18 COVID-19 patients and 34 healthy controls was analyzed. During active COVID-19 disease, neutrophils showed reduced CD10 (p = 0.0360), increased CD11b (p = 0.0002) and decreased CD62L expression (p < 0.0001) compared to healthy controls. During active COVID-19 disease, fNLF stimulated neutrophils showed decreased CD10 levels (p < 0.0001). Three to six months after COVID-19 disease, unstimulated neutrophils showed lowered CD62L expression (p = 0.0003) and stimulated neutrophils had decreased CD10 expression (p = 0.0483) compared to healthy controls. Both (un)stimulated CD10 levels increased 3 to 6 months after active disease (p = 0.0120 and p < 0.0001, respectively) compared to during active disease. Eosinophil blood counts were reduced during active COVID-19 disease and increased 3 to 6 months after infection (p < 0.0001). During active COVID-19, eosinophils showed increased unstimulated CD11b (p = 0.0139) and decreased (un)stimulated CD62L expression (p = 0.0036 and p = 0.0156, respectively) compared to healthy controls. Three to six months after COVID-19 disease, (un)stimulated eosinophil CD62L expression was decreased (p = 0.0148 and p = 0.0063, respectively) and the percentage of CD11bbright cells was increased (p = 0.0083 and p = 0.0307, respectively) compared to healthy controls. Conclusion: Automated flow cytometry analysis reveals specific mature neutrophil and eosinophil activation patterns in primary care patients with COVID-19 disease, during and 3 to 6 months after active disease. This suggests that the neutrophil and eosinophil compartment are long-term affected by COVID-19 in primary care patients. This indicates that these compartments may be involved in the pathogenesis of long COVID.

Chemical tools for epichaperome-mediated interactome dysfunctions of the central nervous system.

Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer's disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.

Ottawa classification for symptomatic acetabular dysplasia assessment of interobserver and intraobserver reliability.

AIMS: The aim of the current study was to assess the reliability of the Ottawa classification for symptomatic acetabular dysplasia. METHODS: In all, 134 consecutive hips that underwent periacetabular osteotomy were categorized using a validated software (Hip2Norm) into four categories of normal, lateral/global, anterior, or posterior. A total of 74 cases were selected for reliability analysis, and these included 44 dysplastic and 30 normal hips. A group of six blinded fellowship-trained raters, provided with the classification system, looked at these radiographs at two separate timepoints to classify the hips using standard radiological measurements. Thereafter, a consensus meeting was held where a modified flow diagram was devised, before a third reading by four raters using a separate set of 74 radiographs took place. RESULTS: Intrarater results per surgeon between Time 1 and Time 2 showed substantial to almost perfect agreement among the raters (κappa = 0.416 to 0.873). With respect to inter-rater reliability, at Time 1 and Time 2 there was substantial agreement overall between all surgeons (Time 1 κappa = 0.619; Time 2 κappa = 0.623). Posterior and anterior rating categories had moderate and fair agreement at Time 1 (posterior κappa = 0.557; anterior κappa = 0.438) and Time 2 (posterior κappa = 0.506; anterior κappa = 0.250), respectively. At Time 3, overall reliability (κappa = 0.687) and posterior and anterior reliability (posterior κappa = 0.579; anterior κappa = 0.521) improved from Time 1 and Time 2. CONCLUSION: The Ottawa classification system provides a reliable way to identify three categories of acetabular dysplasia that are well-aligned with surgical management. The term 'borderline dysplasia' should no longer be used.Cite this article: Bone Joint Res. 2020;9(5):242-249.

Paradigms for Precision Medicine in Epichaperome Cancer Therapy.

Alterations in protein-protein interaction networks are at the core of malignant transformation but have yet to be translated into appropriate diagnostic tools. We make use of the kinetic selectivity properties of an imaging probe to visualize and measure the epichaperome, a pathologic protein-protein interaction network. We are able to assay and image epichaperome networks in cancer and their engagement by inhibitor in patients' tumors at single-lesion resolution in real time, and demonstrate that quantitative evaluation at the level of individual tumors can be used to optimize dose and schedule selection. We thus provide preclinical and clinical evidence in the use of this theranostic platform for precision medicine targeting of the aberrant properties of protein networks.

HSP90-incorporating chaperome networks as biosensor for disease-related pathways in patient-specific midbrain dopamine neurons.

Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors. Through this method we identify STAT3 and NF-κB signaling activation as examples of genetic stress, and phospho-tyrosine hydroxylase (TH) activation as an example of toxic stress-induced pathways in PD neurons. Importantly, pharmacological inhibition of the stress chaperome network reversed abnormal phospho-STAT3 signaling and phospho-TH-related dopamine levels and rescued PD neuron viability. The use of chemical sensors of chaperome networks on hPSC-derived lineages may present a general strategy to identify molecular events associated with neurodegenerative diseases.

Performance of a cylindrical diode array for use in a 1.5 T MR-linac.

At the UMC Utrecht, a linear accelerator with integrated magnetic resonance imaging (MRI) has been developed, the MR-linac. Patient-specific quality assurance (QA) of treatment plans for MRI-based image guided radiotherapy requires QA equipment compatible with this 1.5 T magnetic field. The purpose of this study was to examine the performance characteristics of the ArcCHECK-MR in a transverse 1.5 T magnetic field. To this end, the short-term reproducibility, dose linearity, dose rate dependence, field size dependence, dose per pulse dependence and inter-diode dose response variation of the ArcCHECK-MR diode array were evaluated on a conventional linac and on the MR-linac. The ArcCHECK-MR diode array performed well for all tests on both linacs, no significant differences in performance characteristics were observed. Differences in the maximum dose deviations between both linacs were less than 1.5%. Therefore, we conclude that the ArcCHECK-MR can be used in a transverse 1.5 T magnetic field.

Structure-activity relationship in a purine-scaffold compound series with selectivity for the endoplasmic reticulum Hsp90 paralog Grp94.

Grp94 is involved in the regulation of a restricted number of proteins and represents a potential target in a host of diseases, including cancer, septic shock, autoimmune diseases, chronic inflammatory conditions, diabetes, coronary thrombosis, and stroke. We have recently identified a novel allosteric pocket located in the Grp94 N-terminal binding site that can be used to design ligands with a 2-log selectivity over the other Hsp90 paralogs. Here we perform extensive SAR investigations in this ligand series and rationalize the affinity and paralog selectivity of choice derivatives by molecular modeling. We then use this to design 18c, a derivative with good potency for Grp94 (IC50 = 0.22 µM) and selectivity over other paralogs (>100- and 33-fold for Hsp90α/ß and Trap-1, respectively). The paralog selectivity and target-mediated activity of 18c was confirmed in cells through several functional readouts. Compound 18c was also inert when tested against a large panel of kinases. We show that 18c has biological activity in several cellular models of inflammation and cancer and also present here for the first time the in vivo profile of a Grp94 inhibitor.

Relative dosimetry in a 1.5 T magnetic field: an MR-linac compatible prototype scanning water phantom.

The MR-linac is a hybrid MRI radiotherapy system allowing dose delivery in a 1.5 T magnetic field. This paper presents the design and performance of a prototype MR-linac compatible scanning water phantom. Since a scanning water phantom requires dose detectors, the performance air-filled ionization chambers in the magnetic field was characterized. We have found that the linearity and reproducibility of an ionization chamber are unaffected by the magnetic field. Also, moving the ionization chambers in a magnetic field during irradiation does not affect the dose response. When scanning in-plane profiles, the change in irradiation orientation can influence the ionization chamber dose response by up to 0.4%. However this effect can be eliminated by rotating the ionization chamber by 90° before measuring the in-plane profile. The performance of the total scanning water phantom was validated at a clinical setup in a 0 T magnetic field. There was no significant difference between the dose profiles measured with a standard clinical scanning water phantom and the profiles measured with the MR-linac compatible scanning water phantom. The performance of the MR-linac scanning water phantom in the MR-linac was validated using Gafchromic EBT2 film. There was no significant difference in dose profiles between the MR-linac scanning water phantom and the radiochromic film. These results indicate that automated scanning water phantom measurements using ionization chamber detectors are possible in the MR-linac.

Performance of a multi-axis ionization chamber array in a 1.5 T magnetic field.

At the UMC Utrecht a prototype MR-linac has been installed. The system consists of an 8 MV Elekta linear accelerator and a 1.5 T Philips MRI system. This paper investigates the performance of the IC PROFILER™, a multi-axis ionization chamber array, in a 1.5 T magnetic field. The influence of the magnetic field on the IC PROFILER™ reproducibility, dose response linearity, pulse rate frequency dependence, power to electronics, panel orientation and ionization chamber shape were investigated. The linearity, reproducibility, pulse rate frequency dependence, panel orientation and ionization chamber shape are unaffected by the magnetic field. When the measurements results are normalized to the centre reference chamber, the measurements can commence unaltered. Orientation of the ionization chambers in the magnetic field is of importance, therefore caution must be taken when comparing or normalizing results from several different axes. IC PROFILER™ dose profiles were compared with film dose profiles obtained simultaneously in the MR-linac. Deviation between the film and the IC PROFILER™ data was caused by the noise in the film, indicating correct performance of the IC PROFILER™ in the transverse 1.5 T magnetic field.

Pre-clinical efficacy of PU-H71, a novel HSP90 inhibitor, alone and in combination with bortezomib in Ewing sarcoma.

Ewing sarcoma is characterized by multiple deregulated pathways that mediate cell survival and proliferation. Heat shock protein 90 (HSP90) is a critical component of the multi-chaperone complexes that regulate the disposition and activity of a large number of proteins involved in cell-signaling systems. We tested the efficacy of PU-H71, a novel HSP90 inhibitor in Ewing sarcoma cell lines, primary samples, benign mesenchymal stromal cells and hematopoietic stem cells. We performed cell cycle analysis, clonogenic assay, immunoblot analysis and reverse phase protein array in Ewing cell lines and in vivo experiments in NSG and nude mice using the A673 cell line. We noted a significant therapeutic window in the activity of PU-H71 against Ewing cell lines and benign cells. PU-H71 treatment resulted in G2/M phase arrest. Exposure to PU-H71 resulted in depletion of critical proteins including AKT, pERK, RAF-1, c-MYC, c-KIT, IGF1R, hTERT and EWS-FLI1 in Ewing cell lines. Our results indicated that Ewing sarcoma tumor growth and the metastatic burden were significantly reduced in the mice injected with PU-H71 compared to the control mice. We also investigated the effects of bortezomib, a proteasome inhibitor, alone and in combination with PU-H71 in Ewing sarcoma. Combination index (CI)-Fa plots and normalized isobolograms indicated synergism between PU-H71 and bortezomib. Ewing sarcoma xenografts were significantly inhibited when mice were treated with the combination compared to vehicle or either drug alone. This provides a strong rationale for clinical evaluation of PU-H71 alone and in combination with bortezomib in Ewing sarcoma.

Improved targeting of JAK2 leads to increased therapeutic efficacy in myeloproliferative neoplasms.

The discovery of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) led to clinical development of Janus kinase (JAK) inhibitors for treatment of MPN. These inhibitors improve constitutional symptoms and splenomegaly but do not significantly reduce mutant allele burden in patients. We recently showed that chronic exposure to JAK inhibitors results in inhibitor persistence via JAK2 transactivation and persistent JAK-signal transducer and activator of transcription signaling. We performed genetic and pharmacologic studies to determine whether improved JAK2 inhibition would show increased efficacy in MPN models and primary samples. Jak2 deletion in vivo led to profound reduction in disease burden not seen with JAK inhibitors, and deletion of Jak2 following chronic ruxolitinib therapy markedly reduced mutant allele burden. This demonstrates that JAK2 remains an essential target in MPN cells that survive in the setting of chronic JAK inhibition. Combination therapy with the heat shock protein 90 (HSP90) inhibitor PU-H71 and ruxolitinib reduced total and phospho-JAK2 and achieved more potent inhibition of downstream signaling than ruxolitinib monotherapy. Combination treatment improved blood counts, spleen weights, and reduced bone marrow fibrosis compared with ruxolitinib alone. These data suggest alternate approaches that increase JAK2 targeting, including combination JAK/HSP90 inhibitor therapy, are warranted in the clinical setting.

Towards reference dosimetry for the MR-linac: magnetic field correction of the ionization chamber reading.

In the UMC Utrecht a prototype MR-linac has been installed. The system consists of a 6 MV Elekta (Crawley, UK) linear accelerator and a 1.5 T Philips (Best, The Netherlands) Achieva MRI system. This paper investigates the feasibility to correct the ionization chamber reading for the magnetic field within the dosimetry calibration method described by Almond et al (1999 Med. Phys. 26 1847-70). Firstly, the feasibility of using an ionization chamber in an MR-linac was assessed by investigating possible influences of the magnetic field on NE2571 Farmer-type ionization chamber characteristics: linearity, repeatability, orientation in the magnetic field; and AAPM TG51 correction factor for voltage polarity and ion recombination. We found that these AAPM correction factors for the NE2571 chamber were not influenced by the magnetic field. Secondly, the influence of the permanent 1.5 T magnetic field on the NE2571 chamber reading was quantified. The reading is influenced by the magnetic field; therefore, a correction factor has been added. For the standardized setup used in this paper, the NE2571 chamber reading increases by 4.9% (± 0.2%) due to the transverse 1.5 T magnetic field. Dosimetry measurements in an MR-linac are feasible, if a setup-specific magnetic field correction factor (P1.5 T) for the charge reading is introduced. For the setup investigated in this paper, the P1.5 T has a value of 0.953.

Experimental and structural testing module to analyze paralogue-specificity and affinity in the Hsp90 inhibitors series.

We here describe the first reported comprehensive analysis of Hsp90 paralogue affinity and selectivity in the clinical Hsp90 inhibitor chemotypes. This has been possible through the development of a versatile experimental assay based on a new FP-probe (16a) that we both describe here. The assay can test rapidly and accurately the binding affinity of all major Hsp90 chemotypes and has a testing range that spans low nanomolar to millimolar binding affinities. We couple this assay with a computational analysis that allows for rationalization of paralogue selectivity and defines not only the major binding modes that relay pan-paralogue binding or, conversely, paralogue selectivity, but also identifies molecular characteristics that impart such features. The methods developed here provide a blueprint for parsing out the contribution of the four Hsp90 paralogues to the perceived biological activity with the current Hsp90 chemotypes and set the ground for the development of paralogue selective inhibitors.

Integrated megavoltage portal imaging with a 1.5 T MRI linac.

In this note, the feasibility of complementing our hybrid 1.5 T MRI linac (MRL) with a megavoltage (MV) portal imager is investigated. A standard aSi MV detector panel is added to the system and both qualitative and quantitative performances are determined. Simultaneous MR imaging and transmission imaging can be performed without mutual interference. The MV image quality is compromised by beam transmission and longer isocentre distance; still, the field edges and bony anatomy can be detected at very low dose levels of 0.4 cGy. MV imaging integrated with the MRL provides an independent and well-established position verification tool, a field edge check and a calibration for alignment of the coordinate systems of the MRI and the accelerator. The portal imager can also be a valuable means for benchmarking MRI-guided position verification protocols on a patient-specific basis in the introductory phase.

Raman spectroscopic analysis of isomers of biliverdin dimethyl ester.

The constitutional isomers of biliverdin dimethyl ester, IX alpha and XIII alpha, were studied by resonance Raman spectroscopy. The far-reaching spectral similarities suggest that despite the different substitution patterns, the compositions of the normal modes are closely related. This conclusion does not hold only for the parent state (ZZZ, sss configuration) but also for the configurational isomers which were obtained upon double-bond photoisomerization. Based on a comparison of the resonance Raman spectra, a EZZ configuration is proposed for one of the two photoisomers of biliverdin dimethyl ester IX alpha, while a ZZE, ssa configuration has been assigned previously to the second isomer.

A coherent pattern among social behavior, blood pressure, corticosterone and catecholamine measures in individual male rats.

Behavioral and physiological responses of 18 chronically cannulated male TMD-S3 rats were assessed during various social interactions with conspecifics, both with and without the possibility for physical contact (social vs. psychosocial stimulation). Response magnitudes (behavior, blood pressure, plasma catecholamines) depend upon both the social environmental requirements (offense, defense, psychosocial stimulus following defense) and individual characteristics. The more competitive males generally reacted with higher responses of blood pressure and catecholamines than more passive rats. In addition, these competitive males had higher baseline levels of noradrenaline. The present experiment shows that male rats differ in the individual sympathetic tone and reactivity in relation to their behavior in a social environment.