Here, PEGPH20 response was assessed by multiparametric magnetic resonance imaging (MRI) in three orthotopic breast tumour models. Treatment of 4T1/HAS3 tumours, the model with the highest HA accumulation, reduced T1 and T2 relaxation times and the apparent diffusion coefficient (ADC), and increased the magnetisation transfer ratio, consistent with lower tissue water content and collapse of the extracellular space. The transverse relaxation rate R2* increased, consistent with greater erythrocyte accessibility following vascular decompression. Treatment of MDA‐MB‐231 LM2‐4 tumours reduced ADC and dramatically increased tumour viscoelasticity measured by MR elastography. Correlation matrix analyses of data from all models identified ADC as having the strongest correlation with HA accumulation, suggesting that ADC is the most sensitive imaging biomarker of tumour response to PEGPH20.Hyaluronan (HA) is a key component of the dense extracellular matrix in breast cancer, and its accumulation is associated with poor prognosis and metastasis. Pegvorhyaluronidase alfa (PEGPH20) enzymatically degrades HA and can enhance drug delivery and treatment response in preclinical tumour models. Clinical development of stromal‐targeted therapies would be accelerated by imaging biomarkers that inform on therapeutic efficacy  Diffusion‐weighted MRI and elastography can non‐invasively inform on preclinical breast tumour response to extracellular matrix modulation, specifically hyaluronan degradation by PEGPH20, and may facilitate the clinical development of stromal‐targeted therapies. ADCapparent diffusion coefficientWSCVwithin‐subject test–retest coefficient of variationDABdiaminobenzidineDWIdiffusion‐weighted imagingECMextracellular matrixFBSfoetal bovine serumFFPEformalin‐fixed paraffin‐embeddedFOVfield of viewGAGglycosaminoglycandGelastic moduluslGviscous modulusH&Ehaematoxylin and eosinHAhyaluronanHAS3hyaluronan synthase 3IFPinterstitial fluid pressureIHCimmunohistochemistryIS‐MRIintrinsic susceptibility magnetic resonance imagingMGEmultiple gradient echoMREmagnetic resonance elastographyMRImagnetic resonance imagingMTRmagnetisation transfer ratioPEGPH20pegvorhyaluronidase alfaPTFEpolytetrafluoroethylene2*Rtransverse relaxation rateRARErapid acquisition with refocused echoesROIregion of interestSEMstandard error of the meanSSsolid stressSTRshort tandem repeat1Tlongitudinal relaxation time2Ttransverse relaxation timeTEecho timeTRrepetition timeTTPtotal tissue pressure1N‐acetylglucosamine. HA is overexpressed in 25–30% of human tumours, including breast cancer in which the extent of HA accumulation is associated with metastasis and can predict for overall survival [Malignant primary breast tumours are often characterised by a strong desmoplastic reaction, which results in a dense collagenous stroma with extracellular matrix (ECM) rigidity, providing a permissive scaffold for tumour growth. This stromal‐dense phenotype is associated with tumour progression  and poor drug delivery , 2. Breasurvival , 5, 6. Hsurvival , 8, 9.Numerous stromal targeted treatment strategies and accompanying biomarkers are being developed to alter tumour–stroma interactions for therapeutic gain, including drugs targeted against HA , 10. Pegin vivo. MR image acquisition can (a) be sensitised to numerous independent contrast mechanisms related to the physical state of tissue water and its ability to diffuse and (b) provide a direct measure of the viscoelastic properties of tissue (elastography). Parametric maps can be calculated and used to inform on tumour structure and function in situ.HA degradation (depolymerisation) by PEGPH20 has been associated with a decrease in tumour water content , 9. Thisin vivo [1) and transverse (T2) relaxation times sensitive to the organisation of neighbouring water molecules , the magnetisation transfer ratio (MTR) which informs on the proportion of water bound to macromolecules, the apparent diffusion coefficient (ADC) sensitive to tissue water diffusivity, the transverse relaxation rate (R2*) sensitive to paramagnetic species including deoxyhaemoglobin in haemodynamic vasculature, and elastic (Gd) and viscous (Gl) components of the complex shear modulus that are measures of tumour viscoelastic properties. Pathological correlates from digital pathology of MRI‐aligned tissue sections were also sought. Collectively, the data were used to appraise these MRI biomarkers to (a) inform on HA accumulation in breast cancer models and their response to PEGPH20 and (b) evaluate whether the imaging biomarkers yield additional mechanistic insight into modulation of the ECM.In this preclinical study, we applied a multiparametric MRI strategy, incorporating the use of magnetic resonance elastography (MRE), to characterise orthotopic breast tumours derived from cell lines with differing levels of HA accumulation, and assess their subsequent response to PEGPH20 in vivo . Our mul22.1Mycoplasma infection, and cell identity was authenticated by short tandem repeat (STR) profiling. luc‐MDA‐MB‐231 LM2‐4 highly malignant luciferase‐expressing triple‐negative breast cancer cells isolated from a lung metastasis were provided by Dr R. Kerbel  and cultured in Dulbecco's modified eagle's medium  supplemented with 10% (v/v) foetal bovine serum . 4T1 murine breast adenocarcinoma cells and 4T1/HAS3 murine breast adenocarcinoma cells which overexpress HA synthase 3 (HAS3) were provided under a research collaboration agreement with Halozyme Therapeutics . 4T1 and 4T1/HAS3 cells were cultured in RPMI 1640 medium  supplemented with 10% FBS with the addition of 100 μg·mL−1 hygromycin B  for HAS3 selection in 4T1/HAS3 cells.All cell lines tested negative for 2.2In Vivo Experiments (ARRIVE) guidelines [ad libitum.All animal experiments were approved by the Institute of Cancer Research Animal Welfare and Ethical Review Body, performed in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986, the United Kingdom National Cancer Research Institute guidelines for the welfare of animals in cancer research, and reported according to the Animal Research: Reporting idelines , 16. The5 cells in 100 μL of sterile serum‐free medium into the third mammary fat pad of adult female BALB/c mice . Human luc‐MDA‐MB‐231 LM2‐4 breast tumours were propagated orthotopically in the third mammary fat pad of adult female athymic NCr‐Foxn1nu mice by injecting 2 × 106 cells in 100 μL Matrigel  and serum‐free medium suspension . Tumour development was monitored twice‐weekly using callipers. Tumour volumes reported herein were measured using multi‐slice anatomical T2‐weighted MRI. A total number of 64 mice were used for this research.Murine 4T1 and 4T1/HAS3 breast tumours were propagated orthotopically by injecting 1 × 102.3−1 dose were used [PEGPH20, a PEGylated form of the recombinant human hyaluronidase PH20, was provided by Halozyme Therapeutics and used to enzymatically degrade tumour HA . A 24‐h ere used . PEGPH202.43, respectively . Mice were given 5 h to recover from anaesthesia before administration of PEGPH20 or saline. MRI was then repeated 24 h later.Pre‐treatment MRI was performed when 4T1, 4T1/HAS3 and MDA‐MB‐231 LM2‐4 tumours reached 453 ± 23, 335 ± 23 and 472 ± 24 mmMRI data were acquired on a 7T BioSpec 70/20 USR horizontal MRI system  using a purpose‐built MRE platform, and a 4 cm volume coil positioned at the isocentre of the magnet .−1 (4T1 and 4T1/HAS3) or 9 mL·kg−1 (MDA‐MB‐231 LM2‐4) intraperitoneal injection of Hypnorm™  and Hypnovel™  and sterile water in a 1 : 1 : 2 ratio. Body temperature was maintained at approximately 37 °C using a water heating pad, and breathing rate was monitored using physiological monitoring equipment .For MRI, tumour‐bearing mice were anaesthetised with a 7 mL·kg2‐weighted images were first acquired using a rapid acquisition with refocused echoes (RARE) sequence  and used to determine the tumour volume, plan the subsequent functional MRI acquisition and optimise the local magnetic field (B0) homogeneity over the tumour using a localised map shim.Anatomical T1 and T2 relaxation times (ms), MT‐RARE  to derive the MTR (%), respiratory‐gated diffusion‐weighted images  for determination of the ADC (×10−6 mm2·s−1), intrinsic susceptibility (IS‐MRI) using a multiple gradient echo (MGE) sequence  to estimate R2* (s−1), and finally MRE  to quantify Gd and Gl (kPa). The total acquisition time for all the sequences including MRE was ∼ 45 min.The subsequent multiparametric MRI protocol included the acquisition of inversion recovery (IR)‐TrueFISP images  for estimating the native TThe MRE set‐up was as previously described . An exte1, T2, MTR, ADC and R2* were reconstructed using in‐house software written in IDL from a 1 mm thick axial slice over a 3 × 3 cm FOV taken from the tumour centre [d and Gl were reconstructed isotropically from three 0.3 mm thick axial slices over a 1.92 × 1.92 cm fov using software shared by Prof Ralph Sinkus. Median values for each tumour were calculated from a region of interest (ROI) which contained viable tumour tissue and excluded necrosis. Areas of necrosis were drawn on T2‐weighted images and confirmed by examination of aligned H&E‐stained histological sections.Parametric maps of Tr centre . Paramet2.5in vivo immediately after post‐treatment MRI using a Millar Mikro‐Tip® piezoelectric mouse pressure catheter . To isolate IFP from total tissue pressure, the catheter was covered with modified polytetrafluoroethylene (PTFE) tubing (ID = 0.4 mm) [labchart .Invasive tumour IFP measurements were performed  0.4 mm) . The PTF2.62‐weighted MRI, tumours were carefully dissected and bisected at the MRI plane. One tumour half was weighed, dried in a lyophiliser for 24 h and then reweighed. Tumour water content (%) was calculated using [Guided by the post‐treatment Ted using :Water c2.7The other tumour half was fixed in formalin and embedded in paraffin. Serial formalin‐fixed paraffin‐embedded (FFPE) tissue sections (5 μm) were cut using a microtome . Tinctorial haematoxylin and eosin (H&E) staining was performed for the assessment of nuclear density, morphology and necrosis. HA was detected by an affinity histochemistry assay using a biotinylated, recombinant HA‐binding protein  [definiens tissue studio® . The extent (%), fractal dimension and entropy associated with collagen I & III were quantified at 20× magnification from picrosirius red staining as previously described [Whole‐slide images were digitised using a Nanozoomer XR scanner . Nuclear density, HA (%) and blood vessel density were quantified at 40× magnification from H&E, HTI‐601 and CD31 staining, respectively, using escribed .2.8HA density parametric maps were generated as a visual aid for the spatial comparison of MRI with histology within a single tumour and were not used for any numerical analyses. Whole‐slide images of HA staining were processed to match the MRI resolution (234 × 234 μm). The brown diaminobenzidine (DAB) staining was extracted from HA‐stained whole slide images. Each pixel in the 20× magnification HA‐extracted images had a squared resolution of 0.46 μm, so each MRI voxel corresponded to a 518 × 518 pixel region in the digital histology image. Each pixel in the HA‐extracted image was marked as ‘1’ if it is HA‐positive or ‘0’ if it is HA‐negative. The fraction of pixels occupied by HA was calculated within 518 × 518 pixel regions and that fraction represented a single voxel in the final MRI resolution‐matched image as previously described .2.9prism 9 , and data are presented as mean ± 1 standard error of the mean (SEM). Significant differences were identified using an unpaired Student's t‐test or a one‐way ANOVA with multiple comparisons with a significance level of 5%. A q value is shown in the case of multiple comparisons and is a P‐value which has been adjusted for the false discovery rate (FDR) using the two‐stage set up method by Benjamini, Krieger and Yekutieli. Correlations were evaluated using the Pearson correlation coefficient (r) with a significance level of 5%. Correlation matrices were created using prism 9.Statistical analyses were performed using WS) for T1, T2, MTR, ADC, R2*, Gd and Gl was calculated from repeated 4T1, 4T1/HAS3 and MDA‐MB‐231 LM2‐4 tumour MRI prior to and 24 h after saline using:m is the mean of all the pre‐saline and post‐saline MRI, Δ is the difference between the paired repeats, and n is the number of pairs [The within‐subject test–retest coefficient of variation , determined from the pre‐ and 24 h post‐saline data for all control tumours, were 2.2% (T1), 6.6% (T2), 5.0% (MTR), 16% (ADC), 16% (R2*), 13% (Gd) and 16% (Gl).Representative MRI data acquired from 4T1, 4T1/HAS3 and MDA‐MB‐231 LM2‐4 tumours treated with either PEGPH20 or saline are shown in Fig. 1, T2, MTR, ADC, R2*, Gd and Gl are summarised for each tumour model before and following treatment with saline or PEGPH20 in Table Mean values of MRI‐derived tumour volume, T2*, and significantly reduced T1, T2 and ADC in 4T1/HAS3 tumours. PEGPH20 treatment of MDA‐MB‐231 LM2‐4 tumours significantly decreased ADC, and significantly increased tumour viscoelastic moduli Gd and Gl. The relatively large increase in Gd and Gl following PEGPH20 (between 49% and 163%) occurred in six of the 11 MDA‐MB‐231 LM2‐4 tumours evaluated.PEGPH20 treatment significantly reduced the growth of 4T1, 4T1/HAS3 and MDA‐MB‐231 LM2‐4 orthotopic breast tumours (Table P = 0.03) and 4T1/HAS3 cohorts  and MDA‐MB‐231 LM2‐4 tumours . Saline control MDA‐MB‐231 LM2‐4 tumours contained significantly less collagen than 4T1 (P = 0.003) and 4T1/HAS3 (P = 0.02) tumours. Furthermore, the 2D distribution of collagen in saline control MDA‐MB‐231 LM2‐4 tumours had a significantly lower fractal dimension and higher entropy compared to saline control 4T1 (P = 0.0004) and 4T1/HAS3 (P < 0.0001) tumours. Greater collagen content was apparent in some PEGPH20‐treated tumours, particularly in the MDA‐MB‐231 LM2‐4 cohort, though this was not statistically significant overall. CD31 staining revealed that blood vessel density was highest in the 4T1/HAS3 saline control tumours (370 blood vessels per mm2 tissue), followed by 4T1 tumours (246 per mm2), and lowest in MDA‐MB‐231 LM2‐4 tumours (95 per mm2). PEGPH20 treatment did not elicit any significant effect on blood vessel density in any of the tumour models (P > 0.05).Good spatial correspondence was achieved between MRI and histology of imaging‐aligned tissue sections Fig. . Represen) and P‐values (P) for each correlation are shown in Fig. 2 negatively correlated with MTR in all cases whether the saline and PEGPH20 data were evaluated together or kept separate . T1 and ADC negatively correlated with nuclear density .The clear spatial correspondence between the MRI and histology enabled an assessment of the relationships between the MRI biomarkers and the underlying pathology, both within tumours and between tumours. The between‐tumour correlation matrix determined for all the post‐treatment  measurements is shown in Fig. 2, MTR and ADC . HA accumulation was positively correlated with T2  and negatively correlated with MTR . Although similar linear trendlines were apparent, there were no significant correlations between MRI biomarkers and percent HA when considering the saline data in isolation . As well as having the strongest numerical inter‐tumour association, visual comparison of ADC maps with HA staining images revealed that tumour regions with high ADC also had high HA accumulation and vice versa  model of pancreatic ductal adenocarcinoma (PDAC) [The high avidity of HA for binding water, coupled with the reduction in water content following HA degradation, provide a compelling motivation to exploit multiparametric ingiomas , 27, 28.a (PDAC) . HA‐mediADC is sensitive to the amount and mobility of water molecules and can inform upon various tissue properties, including the presence of macromolecules, cell membrane permeability, the characteristic sizes of water‐filled extracellular spaces and equilibrium of intracellular and extracellular water . In viab2* than 4T1 and MDA‐MB‐231 LM2‐4 tumours, suggesting a more vascularised phenotype, and supported by their relatively high vascular density quantified from CD31 immunohistochemistry. Accordingly, treatment with PEGPH20 increased R2* in the 4T1/HAS3 cohort, consistent with decompression of blood vessels and increased accessibility of deoxygenated (paramagnetic) erythrocytes within the perfused tumour vasculature. Quantitation of tumour R2* as a proxy for blood vessel density may provide a predictive biomarker of PEGPH20 response. Perfusion MRI techniques utilising exogenously administered paramagnetic contrast agents may provide more sensitive approaches to inform on tumour vascular changes following HA degradation. Indeed PEGPH20‐mediated improvements in tumour perfusion/permeability and fractional blood volume have been reported in models of PDAC using dynamic contrast enhanced MRI and susceptibility contrast MRI [IS‐MRI revealed that 4T1/HAS3 tumours exhibited a faster baseline Rrast MRI , 33.d and Gl compared to 4T1 tumours which, coupled with quantitation of HTI‐601 staining, suggests that greater HA accumulation contributes to a lowering of the elastic and viscous moduli. Despite this, HA degradation by PEGPH20 induced no change in viscoelastic moduli of 4T1 and 4T1/HAS3 tumours. It may be that HA accumulation during tumour development is associated with a softer and less viscous phenotype, but the extent of HA itself does not directly contribute to tumour viscoelastic properties. Interestingly, PEGPH20 induced an unprecedented ∼ 80% increase in Gd and Gl in MDA‐MB‐231 LM2‐4 tumours. Using MRE and digital pathology, we have previously shown that collagen is a major determinant of the elevated tumour elasticity and viscosity in MDA‐MB‐231 LM2‐4 tumours [d and Gl following administration of PEGPH20. Accumulation of HA may diminish the contribution of collagen to ECM stiffening in these tumours, which is abrogated by PEGPH20, hence the marked increase in tumour stiffness measured by MRE.There is a strong rationale for incorporating MRE into multiparametric MRI investigations of stromal‐dense cancers to assess the underlying biology and response to drugs that target the ECM. MRE revealed that 4T1/HAS3 tumours had lower G tumours . Herein,increases in tumour stiffness and support further evaluation of Gd and Gl as binary imaging biomarkers of treatment response. Furthermore, Gd and Gl derived from 3D MRE measurements were highly correlated and changed in a very similar way following PEGPH20 treatment. Therefore, if MRE were to be used to evaluate human tumour response to PEGPH20, a single measure of stiffness derived from 2D MRE may likely be sufficient and enhance clinical translation of the method.Despite promising preliminary data, the phase III clinical trial of PEGPH20 in combination with standard‐of‐care chemotherapy (HALO‐109‐301) failed to improve progression free or overall survival of patients with previously untreated HA ‘high’ metastatic PDAC , 35. Thein vivo, the reduction in tumour water content is sustained for at least 120 h [A dense ECM can increase solid stress, elevate IFP, impede drug delivery and promote breast tumour progression and metastasis , 3, 40. st 120 h . The relCorrelation matrices were used to provide a deeper assessment of the relationship of the MRI biomarkers to the underlying histology in the presence and absence of HA. The MRI biomarkers investigated may be sensitive, but not specific to HA. To interrogate their sensitivity to HA, PEGPH20‐treated data were included with the intention to provide ‘low HA’ values. This approach reduced the influence of other tumour characteristics to each correlation, as well as increasing the dynamic range of HA values available. ADC exhibited the strongest positive correlation with per cent HA, reaffirming ADC as the most sensitive biomarker of tumour response to PEGPH20, most likely due to its sensitivity to the reduction in size of the extracellular space and quantity of water molecules able to diffuse. PEGPH20‐induced reductions in ADC of similar magnitude were recently reported from early phase clinical trial data in patients bearing tumours from other anatomical sites including colon, lung and pancreas .The positive correlation between ADC and cellularity has been previously reported in several tumour types, including breast cancer , 43. Inc2 and MTR are other, less specific parameters related to percent HA. Greater HA accumulation results in elevated fluid retention, as well as extracellular expansion, which together regulate the amount of free water, thus increasing T2 and attenuating the effects of MT‐inducing components of the ECM such as fibrillar collagen.Td are no longer sensitive to the same tumour microenvironmental components. Whilst HA degradation increases MTR, as shown by an upward shift in the trendline of the PEGPH20 treated tumours, it does not affect the sensitivity of MTR to collagen deposition. In saline control tumours, Gd positively correlated with collagen content and blood vessel density, reaffirming them as determinants of MRE‐derived tumour elastic properties [d positively correlated with nuclear density instead. These associations suggest that HA degradation affects the integrity of the ECM, reducing the ability of fibrillar collagen and blood vessels to increase tumour stiffness. As a result, the viscoelastic properties of PEGPH20‐treated tumours become more reliant on the cellular network. A summary graphic describing the influence of HA on the breast tumour microenvironment based on these relationships is shown in Fig. The correlation matrices suggest that HA degradation alters the tumour microenvironment such that MTR and Goperties , 44. How5Multiparametric MRI has the potential to non‐invasively inform on prognosis and improve risk stratification for patients with stromal‐dense cancer. Numerous stromal modulating strategies that target the biophysical properties of tumours to improve cancer therapies are being exploited, whose clinical development would benefit from imaging biomarkers that accurately inform on tumour response. The data herein show that ADC is a sensitive biomarker of HA modulation, providing a strong rationale to incorporate DWI to accelerate the development of ECM‐targeted therapies. The study also highlights the potential of MRE to inform on tissue mechanics and enable expedient adaptation of treatment regimes. Whilst the development of PEGPH20 has been halted, other approaches to degrade HA in cancer are being investigated , 48, 49,David Kang is an employee of Halozyme Therapeutics. Barbara Blouw was an employee of Halozyme Therapeutics at the time of the study. David Kang and Barbara Blouw are shareholders of Halozyme Therapeutics.ELR, JCB, YJ, and SPR were involved conceptualization. JL, JCB, and SPR were involved in supervision. ELR, JL, KZ‐P, JKRB, JS, CC, BB, DK, RS, YJ, and SPR were involved in methodology and resources. ELR and JL were involved in data acquisition. ELR and KZ‐P were involved in data analysis. ELR was responsible for manuscript preparation . ELR, JL, KZ‐P, JKRB, JS, CC, BB, DK, RS, JCB, YJ, and SPR were involved in manuscript review.https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/1878‐0261.13437.The peer review history for this article is available at Fig. S1. Multiparametric MRI before and 24‐h after saline.Table S1. Summary of the quantitative volumetric and multiparametric MRI data determined prior to and post‐treatment.Fig. S2. Representative MRI and aligned histology images.Fig. S3. Sample sizes (blue) and P‐values (red) for the correlation matrices of each MRI biomarker and histological marker shown in Fig. 5.Click here for additional data file.


It has recently been shown that torsion can break liquid bridges of viscoelastic fluids, with potential application to their clean and rapid dispensing. However, many commonplace fluids  have more complex thixotropic elastoviscoplastic (TEVP) properties that depend on the imposed stress and the timescale of deformation. Using a commercial thermal paste, we show that liquid bridges of TEVP fluids can also be broken by torsion, demonstrating the applicability of the technique for improved dispensing of real industrial fluids. The liquid bridge breaking mechanism is an elastic instability known as “edge fracture.” Dimensional analysis predicts that the effects of thixotropy and plasticity can be neglected during edge fracture. Simulation using a nonlinear, phenomenological TEVP constitutive model confirms such a prediction. Our work yields new insight into the free-surface flows of TEVP fluids, which may be important to processes such as electronic packaging, additive manufacturing, and food engineering. Thixotropic elastoviscoplastic fluids such as adhesives can store energy elastically and resist extensional deformation. Consequently, a long ligament can form between the nozzle and the substrate when those fluids are dispensed, which may collapse after pinch-off and contaminate the underlying substrate. To overcome this stringing problem, we propose using edge fracture, the sudden indentation of the free surface of an elastic fluid subjected to sufficient torsion. Edge fracture cuts the fluid ligament horizontally without needing to apply any extension. Our work pushes the study of edge fracture, a flow instability that researchers tend to avoid in their experiments, into a new direction by showing how this “undesirable” phenomenon can be harnessed to benefit technological applications.Modern dispensing technology such as direct ink writing  and fuseFor a fluid to be printable, it must demonstrate at least two physical properties. First, the fluid must be able to wet the dispensing substrate so that the printed structure stays stationary as the dispensing nozzle moves. Second, the fluid must have a viscosity that changes in response to stimuli such as the shear rate or temperature. Such a property helps the fluid flow more easily through the dispensing nozzle. More importantly, it enables the dispensed fluid to increase viscosity over orders of magnitude. Consequently, the printed structure can retain shape under its weight over a longer time and be solidified later by cooling, solvent evaporation, chemical cross-linking, or sintering , 3.Unfortunately, the physical properties that render a fluid printable often make the dispensing process inefficient. Because the dispensed fluid tends to resist deformation, as the nozzle retracts, a relatively stable liquid bridge can form between the nozzle and the substrate, whose breakup can significantly slow down the dispensing process. To speed up the process, a common approach is to retract the nozzle over a longer distance and with a higher speed such that there will be more significant capillary and shear stresses to destabilize the liquid bridge. Yet, this reduces the precision as a long capillary tail can form after the liquid bridge pinches off. The capillary tail may then fall randomly onto and contaminate the dispensing substrate or the printed structure. This is often referred to as the stringing (or tailing) problem , 21, 39.thixotropic means that the fluid shows a time-dependent response even under a fixed level of force, as a finite amount of time is required for the highly filled particulate phase in the fluid to form a microstructure or for the microstructure to be destroyed. On the other hand, the term elastoviscoplastic means that the fluid shows rheological responses between a viscous fluid and an elastoplastic solid (For particulate composites (fluids composed of particles suspended in a matrix) like conductive adhesives, surface-mount adhesives, and solder pastes, the stringing problem can be severe due to their material composition , 21 and ic solid . As the ic solid , 23. Furic solid , 47, furVarious strategies have been employed to prevent stringing and improve dispensing quality. The simplest is to introduce a dwell time after dispensing to allow the sagging of fluid before the nozzle retracts and moves to its new position , 21. HowEdge fracture is a flow instability often observed in rotational rheometry when an elastic fluid is subjected to a sufficiently strong shear . The pheWe consider a setup consisting of a TEVP liquid bridge sandwiched between two coaxial circular parallel plates. The top plate can move vertically, modeling the dispensing nozzle. The bottom plate can rotate unidirectionally to apply torsion to the liquid bridge Fig. A.The rheology of the TEVP fluid is characterized using the oscillatory shear test, creep test, shear startup test, two-step shear test, and steady shear test Fig. B–F. The Here,  and Wei .The constitutive model has six material parameters: the thixotropic viscosity ηt=ηpl/−s, which network . When th network . When 0<Although the model does not include an explicit yield stress parameter, a stress plateau can be observed in simple shear as ˙→0 Fig. F. This sMeanwhile, the normal stress differences as a function of the shear rate As The constitutive model, coupled with the mass and momentum conservation equations and suitable initial and boundary conditions, allows the TEVP liquid bridge to be numerically simulated, complementing the analysis of the experimental results. Details of the experimental protocol, material characterization, constitutive modeling, and simulation setup can be found in section “Materials and methods.”First, we show that liquid bridges made of the TEVP fluid exhibit extensional behavior unfavorable to the nozzle retraction method of inducing breakup. For a typical liquid bridge of radius The extensional behavior of the TEVP liquid bridge depends upon the speed s−1 Fig. B, initiaFor To inspect the extensional behavior quantitatively, Fig. s et al.  considerThe stringing problem can be prevented by using torsion instead of an extension to destabilize the liquid bridge. Fig. s−1 Fig. A, at t=0For s−1 Fig. B, at t=2Increasing s−1 Fig. C, at t=0Further increasing s−1 Fig. D, at t=0As mentioned above for Fig. Having described qualitatively how the liquid bridge deforms under different levels of torsion, we next discuss the quantitative features. Fig. For see Fig. . Such anBy decreasing the height  mm Fig. A, the lisee Fig. D. BecausDecreasing  mm Fig. B, fewer Further decreasing  mm Fig. C, the spFig. see Fig. , its quasee Fig. B, even tThe liquid bridge deformation shown in Figs. For a liquid bridge subjected to torsion, the characteristic stress is Regarding the thixotropic effect, recall from Eq. e number , which m/2k2 Eq. , the MneThe problem seems somewhat complicated as five dimensionless numbers are involved. However, such complexity can be reduced by a simple order of magnitude analysis. To obtain an upper bound for Re, we assume that see Fig. ; this giAn important physical insight can be gained by further inspecting the orders of magnitude of All in all, it is evident from the above analysis that the indentation phenomenon is a viscoelastic instability governed by Equivalently, the liquid bridge thinning problem can be analyzed using the Tanner number  torsion . The sil see Eq. . In suchThe power-law-like decay behavior of the liquid bridge radius see Fig.  and the fracture . To inspn et al. , suggestFor s−1 Fig. A, initiaFor the higher see Fig. B–D. TherFor s−1 Fig. B, the tiIn this work, we have considered a setup consisting of a TEVP liquid bridge sandwiched between two coaxial circular parallel plates. The top plate could be translated vertically at a speed of Previous studies , 67 havepensions , 74–76, Although our focus is on the edge fracture of TEVP liquid bridges, the methodology employed can be readily used to understand other phenomena involving TEVP materials, which generally depend on the complex interaction between the inertial, elastic, plastic, capillary, viscous, gravitational, and thixotropic effects. For instance, the constitutive model presented in this study Eqs.  and 2 prIn summary, our results provide new insights toward a better understanding of the free-surface flows of TEVP materials. They will inform new designs of nozzles to dispense complex fluids quickly and cleanly, which are important for various industrial and biotechnology applications. Nozzle designs with a rotatable printhead already exist ; they caThe experimental setup consists of a TEVP liquid bridge sandwiched between two coaxial circular parallel plates of radius  mm Fig. A. The plTo eliminate flow histories and ensure that the liquid bridge is axisymmetric, before each experiment, the liquid bridge is slightly stretched from  in Ref. . All expThe TEVP fluid used is a thermal paste of >50 wt% alumina oxide and 5–10 wt% monocrystalline diamond dispersed in 40–45 wt% methyl silicone . Its non-evaporative and chemically stable natures allow experiments to be performed for an extended period, which is essential for the preshearing and relaxation procedures of the liquid bridge. The density of the paste is 6 mN m−1 . Six rheaka the small amplitude oscillatory shear (SAOS) test, is performed with a strain amplitude of A 40 mm diameter stainless steel parallel plate geometry is used for the oscillatory shear test and creep test. C800-grit sandpapers are fixed with double-faced tape to each plate to prevent sample wall slip. Both amplitude and frequency sweeps are performed for the oscillatory shear test. The amplitude sweep is performed with an oscillation frequency of  instead . Such a A 25 mm diameter stainless steel 0.1 rad cone-partitioned plate geometry is used for the shear startup and two-step shear tests. The geometry is a modification of the conventional cone and plate geometry in which only the central 10 mm diameter of the plate is coupled to the stress transducer. The remaining 15 mm diameter of the fluid sample acts as a guard-ring to protect the measurement area, delaying the effect of edge fracture and rendering rheological measurements at higher shear rates possible. For the shear startup test , the us in Ref. , which pAll model material parameters are estimated by performing simultaneous nonlinear regression  to the SIt is worth mentioning that Eq. The three-dimensional (3D) axisymmetric flow of a TEVP fluid confined between two coaxial circular parallel plates of radius  mm Fig. A is consCylindrical coordinates The fluid is of density where s et al. . The syss et al. . The elepgad042_Supplementary_DataClick here for additional data file.


While Rhinovirus/Enterovirus (RV/EV) infections are common, the clinical characteristics of infections in hospitalized adults are not fully understood.Adults ≥ 50 years of age hospitalized for Acute Respiratory Infections (ARI) or exacerbations of CHF or COPD in two hospitals in Atlanta, GA during the 2018-2019 and 2019-2020 respiratory seasons were offered enrollment. Following informed consent, participants were tested via BioFire® FilmArray® respiratory panels of nasopharyngeal and oropharyngeal swabs (combined), and standard-of-care molecular testing results were also recorded. Subjects were considered positive for RV/EV if any method of testing resulted positive. Baseline characteristics and clinical features were gathered via subject interviews and medical record abstractions. Variables were compared between subjects with RV/EV and two control groups: those negative for all pathogens and those negative for only RV/EV. Participants with RV/EV who had co-infections were excluded from the analysis. Descriptive statistics were performed using SAS v9.4.Of 1429 enrolled participants, 123 (8.6%) were positive for RV/EV, of whom 111 had RV/EV alone. When compared to those negative for all tested pathogens (n=1034), participants with RV/EV more commonly had underlying COPD  and less commonly had CHF  or experienced acute myocardial dysfunction . Participants with RV/EV also more commonly experienced fever , cough , sore throat , chest pain , and dyspnea/respiratory distress  than those negative for all pathogens. Differences between RV/EV positive and negative groups were similar to the all pathogen negative group, with the exception of no significant differences in acute myocardial dysfunction, fever, and COPD in the RV/EV negative group.Among older adults hospitalized with ARIs, CHF, and/or COPD exacerbations, RV/EV was associated with symptoms of both upper and lower respiratory tract infection and was more frequent identified among those with COPD.Elizabeth Begier, M.D., M.P.H., Pfizer: EB is an employee of Pfizer, the sponsor of this study|Pfizer: Stocks/Bonds Robin Hubler, MS, Pfizer, Inc.: Employee|Pfizer, Inc.: Stocks/Bonds Qing Liu, M.S., Pfizer Inc.: Stocks/Bonds Bradford J. Gessner, M.D., M.P.H., Pfizer: I am an employee of Pfizer|Pfizer: Stocks/Bonds Benjamin Lopman, PhD, Epidemiological Research and Methods, LLC: Advisor/Consultant|Hillevax, Inc: Advisor/Consultant Satoshi Kamidani, MD, CDC: Grant/Research Support|Emergent BioSolutions: Grant/Research Support|NIH: Grant/Research Support|Pfizer Inc: Grant/Research Support Nadine Rouphael, MD, Icon, EMMES, Sanofi, Seqirus, Moderna: Advisor/Consultant Evan J. Anderson, MD, GSK: Advisor/Consultant|GSK: Grant/Research Support|Janssen: Advisor/Consultant|Janssen: Grant/Research Support|Kentucky Bioprocessing, Inc.: Safety Monitoring Board|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Moderna: Currently an employee|Moderna: Stocks/Bonds|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant|Sanofi Pasteur: Grant/Research Support|Sanofi Pasteur: Safety Monitoring Board|WCG/ACI Clinical: Data Adjudication Board Christina A. Rostad, MD, BioFire Inc.: Grant/Research Support|GlaxoSmithKline Biologicals: Grant/Research Support|Janssen: Grant/Research Support|MedImmune LLC: Grant/Research Support|Meissa Vaccines, Inc.: RSV vaccine technology|Merck & Co., Inc.: Grant/Research Support|Micron Technology, Inc.: Grant/Research Support|Moderna, Inc.: Grant/Research Support|Novavax: Grant/Research Support|PaxVax: Grant/Research Support|Pfizer, Inc.: Grant/Research Support|Regeneron: Grant/Research Support|Sanofi Pasteur: Grant/Research Support


There are no standardised criteria for the ‘regional’ pericolic node in colon cancer, which represents a major cause of the international uncertainty regarding the optimal bowel resection margin. This study aimed to determine ‘regional’ pericolic nodes based on prospective lymph node (LN) mapping.in vivo measurements of the bowel, the anatomical distributions of the feeding artery and LNs were determined in 2996 stages I–III colon cancer patients who underwent colectomy with resection margin >10 cm at 25 institutions in Japan.According to preplanned The mean number of retrieved pericolic nodes was 20.9  per patient. In all patients except seven (0.2%), the primary feeding artery was distributed within 10 cm of the primary tumour. The metastatic pericolic node most distant from the primary tumour was within 3 cm in 837 patients, 3–5 cm in 130 patients, 5–7 cm in 39 patients and 7–10 cm in 34 patients. Only four patients (0.1%) had pericolic lymphatic spread beyond 10 cm; all of whom had T3/4 tumours accompanying extensive mesenteric lymphatic spread. The location of metastatic pericolic node did not differ by the feeding artery's distribution. Postoperatively, none of the 2996 patients developed recurrence in the remaining pericolic nodes.The pericolic nodes designated as ‘regional’ were those located within 10 cm of the primary tumours, which should be fully considered when determining the bowel resection margin, even in the era of complete mesocolic excision.Japanese Society for Cancer of the Colon and Rectum. Internationally, there is a great deal of uncertainty regarding the optimal resection margin of the bowel in colon cancer surgery. A major reason for this is that there are no standardised criteria for regional pericolic nodes to be resected in colon cancer surgery, and, as per the tumour–node–metastasis classification, all pericolic nodes are treated as ‘regional’. The conventional concept of feeding artery-oriented bowel resection often influences surgeons' readiness to include two or more feeding arteries in the resection specimens. In addition, as a result of the recent prevalence toward performing complete mesocolic excision (CME), the length of the resected bowel is often used as a yardstick for evaluating specimen quality, and wider resection of the bowel is regarded as a hallmark of high-quality colon cancer surgery. However, there is currently no robust evidence for the advantage of wider resection of the bowel.This is the first large-scale, prospective multicentre cohort study to identify and assess metastatic pericolic lymph node distribution in patients with stage I–III colon cancer. The satisfactory number of pericolic lymph nodes examined and the methodology whereby intraoperative markings were used to identify the location of the lymph nodes and feeding arteries ensured high-quality lymph node mapping. Pericolic lymphatic spread beyond 10 cm was rare (approximately 0.1%). All exceptional cases with positive distant pericolic nodes had T3/T4 tumours with massive lymph node metastasis and experienced a high incidence of recurrence; this finding indicates that such distant pericolic spread is a form of systemic, rather than local disease. Furthermore, in 99.8% of patients, one or more feeding arteries were located in the pericolic region within 10 cm of the primary tumour. Moreover, the distribution of the feeding artery did not significantly affect the status of the pericolic lymphatic spread, although it did affect the mesenteric lymphatic spread toward the origin of the feeding artery. Finally, intraoperative identification of the feeding artery was 95% accurate, but this was reduced to 86% in patients with a BMI ≥30.Based on the actual anatomical distribution of pericolic lymph node metastasis and its prognostic implication, the 10 cm-rule is a valid and practical criterion to define regional pericolic nodes. Even in the era of CME, it should be emphasized that surgical procedures intended to extend bowel resection beyond the regional pericolic area do not improve the quality of surgery. This should be fully considered when determining the bowel resection margin in curatively-intended surgery for colon cancer.Lymph node (LN) metastasis is the most common mode of spread in colon cancer, and the oncological outcomes of patients who undergo curative colectomy are greatly influenced by the quality of surgery, specifically that of lymphadenectomy. In most patients, the pericolic nodes, i.e., the LNs located along the bowel and marginal artery, are the starting site of metastasis.For example, in surgical textbooks published in the United States, ‘left partial colectomy’ for tumours located at the descending colon has been introduced as a procedure to resect the transverse colon and sigmoid colon up to the middle of each, as well as all of the sigmoid arteries except for the most distal.This multicentre cohort study conducted by the Japanese Society for Cancer of the Colon and Rectum (JSCCR) prospectively assessed metastatic LN distribution, focusing on the distance from the primary tumour and the anatomical association with feeding artery location (UMIN000030331). The main purpose of this study was to determine the criteria for ‘regional’ pericolic nodes to facilitate the standardisation of colon cancer surgery by ascertaining the optimal length of bowel resection.This study was conducted according to the Declaration of Helsinki and comparable Japanese ethical standards. The study was approved by the Institutional Review Boards of the JSCCR and each participating institution. Written informed consent was obtained from all study participants.This is a multicentre, prospective cohort study. The pre-determined target sample size of this study determined on the basis of the consensus of opinion among the investigators in terms of the validity of sub-population analysis according to T stage was 3000, and a total of 3102 patients were enrolled in this study to examine the anatomical distribution of metastatic LNs . After eThe incidence of recurrence associated with the length of the bowel resected was investigated in these 2996 patients using surveillance information in January 2020. The median follow-up period was 44 (0–76) months. A total of 300 patients (10.0%) developed recurrent disease , and the 3-year relapse-free survival (RFS) rate was 88.5% as calculated by the Kaplan–Meier method.Bowel resection was performed to include all pericolic LNs defined as ‘regional’ according to both the 10-cm rule  and the in vivo measurements of the bowel. More specifically, before resecting the bowel, in the natural state with no external tension, points were marked respectively on the bowel  by serosal sutures at 3, 5, 7 and 10 cm from the proximal and distal edges of the tumour. The distal end of the primary feeding artery was also marked with a stitch during the operation.The anatomical location of the feeding artery and LNs were determined based on preplanned After resecting the bowel, the primary feeding artery  was identified in fresh surgical specimens and its anatomical location was recorded . The disJapanese Classification of Colorectal Carcinoma issued by the JSCCR  determined by the intraoperative marking stitches on the bowel , (3) 3 < D ≤ 5 cm , (4) 5 < D ≤ 7 cm , (5) 7 < D ≤ 10 cm  and (6) D > 10 cm .LNs were retrieved from the resected surgical specimens and categorised as pericolic, intermediate, or main LNs based on the LN grouping system of the he JSCCR .9,10 Thehe bowel  as folloThe procedures that were performed to identify the feeding/marginal arteries and to retrieve LNs were exclusively performed by surgeons. LNs were pathologically examined in routine diagnostic practice at each institution with the conventional method with hematoxylin and eosin staining for a representative section of the LN. Tumor nodules with no pathological evidence of LN structure are considered as LN metastases, irrespective of their size and contour morphology.https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000034640).The study endpoints were to determine the anatomical distribution of metastatic LNs, and the incidence of recurrence associated with insufficient lengths of bowel resection  and t-test were used for continuous variables and the chi-square test or Fisher's exact test was used for categorical variables to assess differences in the number of LNs retrieved/involved, feeding artery distribution, accuracy of intraoperative identification of the feeding artery and recurrence rates. Test of Spearman's rank correlation coefficient and Cochran-Armitage trend test were used to assess associations between the location of the primary feeding artery and pericolic tumour spread or tumour spread along supplying arteries. The RFS (time from surgery to the first recurrence or death) was estimated using the Kaplan–Meier method. Alive patients, including those who were lost to follow-up, were censored at their last follow-up date. All statistical analyses were performed using R version 3.6.1  by an experienced mathematical statistician (TN).This study was supported by the Fund of the Japanese Society for Cancer of the Colon and Rectum (JSCCR). No pharmaceutical company nor other agency were involved in this study and this research received no specific grant funding with no role of the funding source in this work.The anatomical distribution of the pericolic LNs is shown in in vivo marking stiches at 3, 5, 7 and 10 cm from the primary tumour, the incidence of LN metastasis was highest in the primary tumour region (27.2%) and decreased with increasing distance from the primary tumour (P = 0.41). The incidence of metastasis in LNs located ≥7 cm from the primary tumour was <1% both proximal and distal to the primary tumour. The incidence according to the T-stage is shown in Overall, 1044 (34.8%) out of 2996 patients had pericolic LN metastasis. Among the 11 pericolic segments made according to the y tumour . There wD ≤ 3 cm segment in 309 patients (10.3%), 3 < D ≤ 5 cm segment in 130 patients (4.3%), 5 < D ≤ 7 cm segment in 39 patients (1.3%) and 7 < D ≤ 10 cm segment in 34 patients (1.1%). Only four patients (0.1%) had LN involvement within >10 cm of the primary tumour.The metastatic LNs were confined within the primary tumour region in 528 patients (17.6%) . The mosFour cases with pericolic LN involvement >10 cm from the primary tumour were reported from different institutions. All had extensive LN metastasis, defined as the involvement of ≥10 LNs and/or main LN involvement .Although the anatomical distribution of positive LNs was associated with the T stage, only 2 (0.1%) of 1443 patients with T3 tumours and 2 (0.4%) of 449 patients with T4a tumours had positive pericolic nodes located >10 cm from the primary tumour . SimilarAmong 2996 patients, a feeding artery flowed into the pericolic region within 10 cm of the primary tumour in 2989 (99.8%), and 1828 patients (61.0%) had a feeding artery that flowed into the primary tumour region . In fourThe distance of the primary feeding artery from the primary tumour differed significantly depending on the location of the primary tumour . The feeDespite a significant difference in the distribution of the primary feeding artery according to the location of the primary tumour, the anatomical distribution of the positive pericolic LNs was not significantly affected by the location of the primary feeding artery . In contP < 0.0001) . The accuracy of the intraoperative identification of the feeding arteries was significantly associated with BMI ( 0.0001) .Table 5ANone of the included patients experienced recurrence in the pericolic LN, although we did observe anastomotic recurrence, likely due to tumour implantation at the anastomotic site, in six patients (0.2%) during the observation period. The recurrence-free survival at 3 years was calculated according to the location of the most distant metastatic pericolic node as 80.4%, 79.1%, 76.2%, 62.7%, and 69.7% for patients with the most distant metastatic pericolic node within the primary tumour, within 3 cm of the primary tumour, within 3–5 cm, within 5–7 cm, and within 7–10 cm from the primary tumour, respectively . Of the in vivo measurement. Based on the actual anatomical distribution of pericolic LN metastasis and its prognostic implication, we concluded that the 10 cm-rule is a valid and practical criterion to define ‘regional’ pericolic nodes.This is the first multicenter cohort study that prospectively assessed LN distribution in terms of the distance from the primary tumor and the anatomical association with feeding artery location according to the preplanned protocol with in vivo measurement of the bowel given that there is a remarkable difference in the length of colorectal segments between in vivo and in vitro. In deed, Goldstein et al.in vivo length following removal from the patient and after being left in an unfixed state for 10–20 min, and by up to 57% after fixation. In previous LN mapping studies,,The mean number of retrieved LNs was 30.8 per patient, which was much higher than figures reported by previous studies, which ranged from 11 to 14 in studies with large cohorts of stage II and III colorectal cancer patients.Both the number of the pericolic nodes retrieved and the number of involved nodes decreased as the distance from the primary tumour increased. Notably, only approximately 1% or less metastatic foci were pathologically identified in pericolic nodes located >10 cm from the primary tumour, irrespective of the T-stage. Some previous studies in the late 1900s using the clearing technique reported cases of distant pericolic nodal metastasis, e.g., 37 cm from the primary tumour in Grinnel's study,,en bloc removal of the mesocolon harbouring metastatic LNs, with sharp anatomic dissection along the embryologic planes and preservation of the intact visceral fascia of the mesocolon,In Western countries, the length of bowel resection is thought to depend on the removal of the colon's arterial supply,General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum and Anus) was published in September 1977.In Japan, the 10-cm rule had been used as the criterion to classify pericolic nodes since the first edition of the Japanese staging manual for colorectal cancer  published on March 2006,In contrast, the The present study shows that the distribution of the feeding artery differs depending on the location of the primary tumour. In general, the arcade of feeding arteries is wide in the transverse and descending colon, especially the splenic flexure site, as compared with the cecum and sigmoid colon.In addition, intraoperative identification of the feeding artery is not always accurate, especially in obese patients. According to our results, in over one in ten patients with a BMI ≥30, surgeons were unable to accurately identify the feeding artery location intraoperatively. This also indicates that the bowel resection policy based on the feeding-artery-oriented criteria for regional pericolic nodes may be suboptimal when standardising colon cancer surgery, and it may be even more so when it comes to the Caucasian European/American population with high BMI.Unlike the situation in pericolic tumour spread, the location of the feeding artery impacted the incidence of central lymphatic spread along the supplying arteries. Specifically, incidences of tumour spread along the supplying arteries were significantly higher in patients with a primary feeding artery distributing in the pericolic region of the primary tumour. In addition, the incidence was lower in patients with a tumour located at the splenic flexure, where feeding arteries are sparsely located, than those with tumours at other sites. Namely, when there is a feeding artery in close proximity to the tumour, tumour cells may easily travel to the main LNs via the network of lymphatic vessels around the feeding artery, indicating the requirement for central vascular ligation achievement.,,We identified seven exceptional cases with no feeding arteries located within 10 cm of the primary tumour. None of them had LN involvement in the pericolic nodes >10 cm from the primary tumour, intermediate LNs or main LNs. These results suggest that segmental resection of the colon with a 10-cm resection margin  and will allow us to determine whether the above findings are ubiquitously observed and can be generalised in patients with colon cancer in other countries.In conclusion, the distribution of the feeding artery did not impact the status of pericolic lymphatic spread, although it might affect that of central lymphatic spread along the supplying arteries that independently impairs survival outcomes.H.U., K.H., and K.S. devised the initial concept and design of the study. A.S., M.S., Ma.I., H.U., T.S., Y.H., T.K., Yus.K., H.I., K.M., K.Y., Ko.K,. K.T., Yuk.K., H.O., M.O., T.M., Ya.T., A.I., J.W., Yu.T., H.S., Ke.K., Y.A., and Mi.I. were responsible for data curation and verification of the raw data. T.N. analysed the data. All authours were involved in data interpretation and validation. H.U. wrote the original draft of the paper and produced the figures and tables of the manuscript. K.S., K.H., A.S., M.S., Ma.I., T.S., Y.H., T.K., Yus.K., H.I., K.M., K.Y., Ko.K., K.T., Yuk.K., H.O., M.O., T.M., Ya.T., A.I., J.W., Yu.T., H.S., Ke.K., Y.A., Mi.I. and T.N. were involved in reviewing and editing drafts of the paper and approving the manuscript. H.U. was involved in the project administration and KS played a role in the funding acquisition, supervision, and decision to submit the manuscript for publication.The data contains personal information, and the study participants have not consented to public data sharing. Data access requires permission from the Japanese Society for Cancer of the Colon and Rectum.All authors declare no competing interests.


BICSTaR is an ongoing, multinational, observational cohort study evaluating the real-world effectiveness and safety of B/F/TAF in antiretroviral therapy (ART) treatment-naïve and treatment-experienced (TE) people with HIV.This analysis of BICSTaR included TE virologically suppressed people with HIV who had started B/F/TAF in clinical practice with/without present/past evidence of HIV drug primary resistance mutations (PRMs). All had viral load (VL) data at baseline (BL). We report virologic and other outcomes at 12 months (M).Table 1).In the overall population, the most common ARTs taken immediately before B/F/TAF were E/C/F/TAF (27%), DTG+F/TAF (9%) and ABC/DTG/3TC (8%). BL genotypic drug resistance testing data were available for 441/996 (44%) participants (ppts); most tests were historic and performed > 60M before starting B/F/TAF  had present/past evidence of PRMs: 13% to NRTI, 11% to NNRTI, 6% to PI, 0.2% to INSTI. The most common PRMs were M184V/I (39 [37%]), ≥ 1 thymidine analog mutation , K103N/S in reverse transcriptase (23 [22%]) and M46I/L in protease (13 [12%]). Primary resistance to > 1 ART drug class was observed in 40 (38%) ppts with PRMs. Ppts with preexisting PRMs were older (≥ 50 years), had more prior ARTs and more prior virologic failure, and had a longer time between HIV diagnosis and starting B/F/TAF versus those without PRMs.Table 2.At 12M, effectiveness  was maintained in 78 (99%) and 739 (98%) ppts with, versus without, any BL PRMs, respectively; 32/33 (97%) of those with M184V/I alone; 13/13 (100%) with M184V/I + 1–2 TAMs; and 2/2 (100%) with M184V/I + ≥ 3 TAMs at BL. No treatment-emergent PRMs to B/F/TAF were reported. Drug-related adverse events (DRAEs) occurred in 17 (16%) ppts with PRMs versus 113 (13%) without PRMs; serious DRAEs occurred in 2 ppts, both had PRMs at BL. Overall, 98/996 (10%) ppts switched from B/F/TAF to other ARTs . Additional outcomes are shown in After 12 months, virologically suppressed people with HIV initiating B/F/TAF in routine clinical practice maintained high rates of effectiveness despite the presence of PRMs (including M184V/I).Benoit Trottier, MD, Gilead: Advisor/Consultant|Gilead: Honoraria|Merck: Advisor/Consultant|Merck: Honoraria|ViiV: Advisor/Consultant|ViiV: Honoraria Fabrice Bonnet, PhD, Gilead: Grant/Research Support|Gilead: Honoraria|Gilead: Educational Miguel Garcia-Deltoro, MD, PhD, AbbVie: Advisor/Consultant|AbbVie: Grant/Research Support|Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Janssen: Board Member|Janssen: Grant/Research Support|MSD: Advisor/Consultant|MSD: Grant/Research Support|ViiV: Advisor/Consultant|ViiV: Grant/Research Support Massimo Andreoni, MD, Gilead: Advisor/Consultant|Moderna: Advisor/Consultant|MSD: Advisor/Consultant Marta Boffito, MD, PhD, FRCP, AstraZeneca: Honoraria|ATEA: Advisor/Consultant|ATEA: Honoraria|Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Gilead: Honoraria|GSK: Advisor/Consultant|GSK: Grant/Research Support|Moderna: Grant/Research Support|MSD: Advisor/Consultant|MSD: Grant/Research Support|MSD: Honoraria|Novavax: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Roche: Advisor/Consultant|Roche: Grant/Research Support|Valneva: Grant/Research Support|ViiV: Advisor/Consultant|ViiV: Grant/Research Support Berend J. van Welzen, MD, PhD, Gilead: Advisor/Consultant|Gilead: Grant/Research Support|ViiV: Advisor/Consultant Dan Turner, MD, Gilead: Advisor/Consultant|Gilead: Honoraria|GSK: Advisor/Consultant|GSK: Honoraria Dai Watanabe, MD, PhD, Gilead Sciences K.K.: Honoraria|Janssen Pharmaceutical K.K.: Honoraria|MSD K.K.: Honoraria|ViiV Healthcare K.K.: Honoraria Alper Gündüz, MD, Gilead: Advisor/Consultant|Gilead: Honoraria|GSK: Advisor/Consultant|GSK: Honoraria|MSD: Advisor/Consultant|MSD: Honoraria David Thorpe, PhD, Gilead: Employment|Gilead: Stocks/Bonds Michelle L. D’Antoni, PhD, Gilead: Employment|Gilead: Stocks/Bonds Tali Cassidy, PhD, Gilead: Employment|Gilead: Stocks/Bonds Andrea Marongiu, PhD, Gilead: Employment|Gilead: Stocks/Bonds Amy R. Weinberg, DNP, MS, Gilead: Employment|Gilead: Stocks/Bonds Richard Haubrich, MD, Gilead: Employment|Gilead: Stocks/Bonds Stefan Scholten, MD, AbbVie: Advisor/Consultant|AbbVie: Honoraria|Cepheid: Grant/Research Support|Cepheid: Honoraria|Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Gilead: Honoraria|Gilead: Congress and travel support|GSK: Grant/Research Support|Janssen: Advisor/Consultant|Janssen: Honoraria|Janssen: Congress support|ViiV: Advisor/Consultant|ViiV: Grant/Research Support|ViiV: Honoraria|ViiV: Congress and travel support


Varicella-Zoster virus (VZV) vasculopathy occasionally occurs in immunocompromised patients and is difficult to treat. The risk factor and optimal therapy remain unclear. Patients with human immunodeficiency virus (HIV) and dysphagia or difficulty in oral intake receive antiretroviral therapy (ART) suspension. However, there remains little evidence regarding ART suspension.We experienced a case of a 55-year-old man diagnosed with HIV and severe multiple cerebral infarctions due to VZV vasculopathy. We started on bictegravir/tenofovir alafenamide/emtricitabine (BIC/TAF/FTC) and acyclovir (ACV), and prednisone. He was started on BIC/TAF/FTC suspension because of deteriorated swallowing. The HIV viral load was increased; however, no drug-resistance genes were detected. We successfully treated him with doltegravir/abacavir/lamibudine suspension. We performed two literature reviews of the administration of BIC/TAF/3TC suspension and VZV vasculopathy in patients with HIV. Three cases of BIC/TAF/3TC suspension were considered treatment failures. Recent history of VZV infection and a CD4 count under 200 μL may be risk factors for VZV vasculopathy. The effective treatment may be using steroid and ACV; however, treatment duration could differ.BIC/TAF/FTC suspension administration may be unstable, and treating ACV and steroid may be optimal therapy for VZV vasculopathy; however, the evidence level is low.The online version contains supplementary material available at 10.1186/s12981-023-00547-7. Bictegravir (BIC) can be administered as a single-tablet regimen and in combination with emtricitabine (FTC) and tenofovir alafenamide (TAF). BIC/FTC/TAF is among the most frequently prescribed antiretroviral therapy (ART), given its simplicity, good safety profile, and high genetic barrier to resistance . HoweverVaricella-zoster virus (VZV) causes chickenpox. The virus then becomes latent in ganglionic neurons. With higher age or an immunocompromised host, the VZV reactivates to cause herpes zoster (shingles). VZV causes severe neurological complications—such as vasculopathy, meningoencephalitis, spinal encephalitis, myelopathy, and postherpetic neuralgia—in immunocompromised hosts, such as patients with Human Immunodeficiency Virus (HIV) . In the We present a patient with HIV and severe VZV vasculopathy who presented with increased HIV viral load and worsened clinical course after BIC/FTC/TAF suspension administration. We performed two literature reviews of the administration of BIC/TAF/3TC suspension and VZV vasculopathy in patients with HIV to collect more evidence.5/mL; cryptococcal antigen, negative; and no ART-resistant HIV mutation. The cerebrospinal fluid (CSF) test findings were as follows: initial pressure, 15 cmH20; cell count, 124 μL (mononuclear cells: 88.7%); protein levels, 388 mg/dL; glucose levels, 87 mg/dL; VZV polymerase chain reaction, 9.4 × 104 copies/mL; and cryptococcal antigen, negative. The CSF culture was negative. Contrast-enhanced magnetic resonance imaging (MRI) of the brain revealed cerebral infarctions in the subcortical areas ; blood glucose level, 153 mg/dL; HIV viral load, 2.4 × 10We reviewed BIC/TAF/FTC suspension and VZV vasculopathy in patients with HIV using database records  and extracted information from these articles  of developed viral mutations. In the literature on VZV vasculopathy in patients with HIV, the mortality was higher before 1999 (29 cases) than after 2000 (48 cases) (79% vs. 21%). This may be because, before ART introduction, VZV diagnosis was mostly based on autopsy. Our analysis of the literature since the year 2000 shows that the median age (range): 35.5 (5–62) years; male: 65%; CD4(/μL) (median 56.5), distribution: > 200, 15%; 100 < CD4 ≤ 200, 20%; ≤ 100, 65%: a recent history of VZV; no VZV infection or more than 1 year, 46%, on admission, 21%; within 3 months, 18%; between 3 and 6 months, 8%; between 6 months and 1 year, 8%: treatment in neurological symptom improvement or stable; ACV + steroid, 82%; ACV alone, 65%: duration of treatment, excluding death; within 2 weeks, 4 cases; 3–4 weeks; 5 cases; > 1 month, 9 cases: outcome; improved neurological findings, 27 cases; residual neurological deficit, 2 cases: death, 10 cases)  [This article presents a patient with HIV who had severe multiple cerebral infarction due to VZV vasculopathy and failed treatment with BIC/TAF/FTC suspension. Although the manufacturer’s labeling indicates the safety of BIC/TAF/FTC suspension , there aIn general, patients with herpes zoster are known to have significantly higher cerebral infarction within 1 year . From ouPatients with HIV with CD4 counts under 200 μL during or after treatment for herpes zoster should be followed carefully for neurological symptoms. DTG/ABC/3TC may be considered over BIC/TAF/FTC in the case of suspension administration.Additional file 1: Table S1 Table showing the process of the literature review regarding crushed BIC/TAF/FTC. Table S2 Table showing the process of the literature review regarding VZV vasculopathy with HIV patient. Figure S1. The process of the literature review regarding crushed BIC/TAF/FTC. Figure S2. The process of the literature review for Varicella-zoster virus vasculopathy with stroke in patients with HIV


T1 relaxometryis a newtechnique that allows nanoscale magnetic resonance measurements. Herewe present its first application in patient samples. More specifically,we demonstrate that relaxometry can determine the free radical loadin samples from arthritis patients. We found that we can clearly differentiatebetween osteoarthritis and rheumatoid arthritis patients in both the synovial fluid itself and cells derived from it. Furthermore,we tested how synovial fluid and its cells respond to piroxicam, acommon nonsteroidal anti-inflammatory drug (NSAID). It is known thatthis drug leads to a reduction in reactive oxygen species productionin fibroblast-like synoviocytes (FLS). Here, we investigated the formationof free radicals specifically. While FLS from osteoarthritis patientsshowed a drastic decrease in the free radical load, cells from rheumatoidarthritis retained a similar radical load after treatment. This offersa possible explanation for why piroxicam is more beneficial for patientswith osteoarthritis than those with rheumatoid arthritis.Diamond-based  Multiplecauses of arthritis require different treatment strategies. The twomost common types of arthritis are osteoarthritis (OA) and rheumatoidarthritis (RA). OA is the most common joint disease worldwide, affectingan estimated 10% of men and 18% of women over 60 years of age.5 Several methods can be utilizedfor measuring radicals, the damage they cause,4 or certain responses to them at the DNA, RNA, or protein level.8 These methods are usually destructive and do not offer spatialresolution. There are also probes that convert into fluorescent moleculeswhen they react with ROS.9 However, thesereactions are irreversible. Thus, the probes reveal the sample’shistory rather than the current state and bleaching limits their usability.10 Spin labels are another alternative for detectingradicals.12 They react with the radicals to form a stablederivative, which can be detected with conventional magnetic resonanceimaging (MRI) or electron spin resonance (ESR). While these methodsare widely used and even allow in vivo imaging, they are limited insensitivity. As a consequence, they typically offer a spatial resolutionin the millimeter range or down to a micrometer range at best.13Oxidative stressis caused by a variety of molecules, and especiallyfree radicals (molecules with an unpaired electron) are challengingto detect.14 Since optical signals canbe read out more sensitively than magnetic signals, the techniqueallows nanoscale magnetic resonance measurements. This technique isso sensitive that even the small magnetic signal of single electronsor a few protons can be detected.17Diamond magnetometry is a method thatmay offer a solution to someof these issues. The technique is based on a fluorescent defect ina diamond, which changes its optical properties based on its magneticsurrounding.T1 relaxometryhas the advantage that it requires onlyoptical excitation and readout. T1 relaxometryhas already been successfully used in physics.20 This methodis sensitive to spin noise and is thus particularly suited to sensingradicals. This was demonstrated for studying aging in yeast cells21 and sensing mitochondrial activity in muralmacrophages22 as well for studying freeradical generation in cells which are impacted by viruses23 or bacteria.24 Herewe explore the applicability of T1 relaxometryfor measuring free radicals in clinical practice. We applied thismethod for the first time in samples from patients.A specific typeof diamond magnetometry called Synovial fluid (SF) was collectedfrom six patients with OA and six patients with RA undergoing primarytotal knee arthroplasty (TKA). Synovial fluid was extracted from thesuprapatellar pouch prior to arthrotomy using a 10 mL syringe anda 16g needle. After harvesting, the sample was transferred to a sterile120 mL sample container , TheNetherlands) within the sterile area of the operation room. The samplewas kept at room temperature until the end of the operation. Later,the sample was placed in an incubator under sterile conditions fora maximum of 30 min before the experiments.T1 relaxometrymeasurements. We excite NV centers with a laser pumping the NV centersinto the bright ms = 0 state of the ground state. Over time the NVcenters relax back into the darker equilibrium between the ms = 0and ms = ±1 states. This process, which we can follow by collectingthe fluorescence intensity, is faster in the presence of radicals.To measure free radical generation,we performed a sequence of 25 and in the Supporting Information.These measurements were performed with a home-built diamond magnetometerwhich is commonly used in the field and described in a previous studyThe workflow of the performed experiments is shown in T1 measurements in a dry state for 30 min asan initial condition. Then we added 200 μL of complete synovialfluid  and measured T1 for another 30 min for the same particle.Then, we added piroxicam to the synovial fluid at a final concentrationof 2 μg/mL and performed T1 measurementsfor 30 min. Control measurements were performed to evaluate the interactionof FNDs with piroxicam and, therefore, its potential impact on therecorded T1 values. This T1 sequence was measured similarly as previously described,except that synovial fluid was replaced with PBS. Measurements incells are described and shown in the Supporting Information.Prior to the measurements, the suspensionof FNDs was placed on 35 mm diameter Petri dishes (suitable for microscopy)and left to dry at RT, resulting in immobilization of nanoparticlesat the dish surface. For every selected FND, we performed All data are reported as mean value ±standard deviation (SD) with at least three independent repetitions.A statistical analysis of data was conducted using GraphPad Prism8.0.1 software, and the significance was tested by a one-way ANOVAand Wilcoxon test.T1 after addingsynovial fluid . In this case, wedid not observe any changes in T1.Osteoarthritis and rheumatoid arthritis arecharacterized by different levels of inflammation with a higher presenceof immune cells for the latter. However, we do not know if that meansthat we can expect their elevated radical level in the synovial fluidof arthritis patients or only in the cellular compartments. When wecompare the changes in luid see 2 to a diFigure S3 in the SupportingInformation, we observed trends in cells similar to those we haveseen in the synovial fluid. The literature has reported that piroxicam affects the production of reactive oxygenspecies by phagocytes.26 Piroxicam is moreeffective in treating osteoarthritis than rheumatoid arthritis.27 The fact that piroxicam reduces radical formationonly in osteoarthritis samples might explain why. We observe the sametrend in the conventional ROS assay. While the two methods providesimilar results in this case, there are a few differences. Our techniquecan measure the current radical load while ROS probes reveal the historyof the sample. It is possible to perform measurements on the sameparticle and location before and after an intervention. This way,a sample can function under its own control. This means that we candifferentiate between the initial variability between cells and patientsand the impact by the intervention. Since FNDs are only sensitiveto radicals in their immediate surroundings, it is possible to obtainlocalized information from within a few nanometers from the particlesurface.We also performed measurements on synovial-fluid-derivedcells.As shown in Supporting Information).We performed flow cytometryexperiments in order to identify which cell types we derived fromOA and RA synovial fluid  and the formyl peptidereceptor-like 1 (FPRL1). They showed that piroxicam inhibits the neutrophilresponses triggered through FPR but not through FPRL1. The inhibitionmechanism was due to a reduced binding of the activating ligand toits cell surface receptor.29 As the formylpeptide receptor (FPR) is also present in the fibroblast-like synoviocytes,we speculate that cells derived from the OA and RA synovial fluidrespond differently to piroxicam due to the expression of differentvariants of FPR receptors.De Sousa et al. have demonstrated that the mitochondrialactivityof the OA and non-OA cells increases when exposed to OA, but not tonon-OA synovial fluid.Here we demonstrate that diamondrelaxometry can be used to correlateclinical efficacy with radical formation after treatment with Piroxicam.We only see a decrease in radical formation in osteoarthritis sampleswhere the drug is more effective compared with samples obtained fromthe patients suffering from rheumatoid arthritis.


The present study aimed to develop a simple dosing score when starting the cardiac glycoside digitoxin in heart failure with reduced ejection fraction (HFrEF) employing first data from the randomized, double-blinded DIGIT-HF trial.2, and BMI < 27 kg/m2 each were assigned one point for the digitoxin dosing score. A score of ≥ 1 indicated the need for dose reduction with sensitivity/specificity of 81.6%/49.7%, respectively. Accuracy was confirmed in a validation data set including 64 patients randomized to digitoxin yielding sensitivity/specificity of 87.5%/37.5%, respectively.In DIGIT-HF, digitoxin was started with a dose of 0.07 mg once daily (o.d.) in all patients. For score derivation, 317 patients were analyzed who had been randomized to digitoxin. In these patients, after scheduled determination of serum levels at study week 6, the digitoxin dose had remained unchanged or had been reduced to 0.05 mg o.d. (97% of patients) to achieve serum concentrations within a predefined range (10.5–23.6 nmol/l). In logistic regression analyses, sex, age, body mass index (BMI), and estimated glomerular filtration rate (eGFR) were associated with need for dose reduction and, therefore, selected for further developing the dosing score. Optimal cut-points were derived from ROC curve analyses. Finally, female sex, age ≥ 75 years, eGFR < 50 ml/min/1.73 m2, BMI < 27 kg/m2, or age ≥ 75 years. In any other patient, digitoxin may be safely started at 0.07 mg o.d.In patients with HFrEF, treatment with digitoxin should be started at 0.05 mg o.d. in subjects with either female sex, eGFR < 50 ml/min/1.73m Further, current guidelines recommend cardiac glycosides for rate control in atrial fibrillation (AF) in patients with and without heart failure [Cardiac glycosides represent a valuable therapeutic option for patients with heart failure and a reduced ejection fraction (HFrEF), who remain symptomatic despite optimized guideline-directed pharmacotherapy and device therapy . Further failure , 3. Howe failure . In addiBoth digoxin and digitoxin are approved cardiac glycosides and most frequently used for the treatment of patients with HFrEF and/or AF. Information about the relationship of cardiac glycoside dose and blood concentration in HFrEF and/or AF based on clinical trial data is only available for digoxin , 5, but The DIGIT-HF trial is a randomized, double-blind placebo-controlled trial investigating the impact of digitoxin on mortality and hospitalizations for worsening heart failure in patients with advanced HFrEF . ImportaThe DIGIT-HF trial is a multicenter, randomized, double-blind, placebo-controlled investigator initiated clinical trial designed to demonstrate the efficacy of digitoxin in patients with HFrEF. Details on the design of the DIGIT-HF trial have been provided elsewhere . Digitoxnanalysis = 317 patients recruited until June 2019) and a validation set . Patients were grouped by the necessity of a dose reduction. Specifically, patients continuing the initial dose of 0.07 mg o.d., because of a digitoxin serum level ≤ 23.6 nmol/l , were compared with patients receiving a dose reduction to 0.05 mg o.d. because of a digitoxin serum level > 23.6 nmol/l or even the recommendation of discontinuation due to a digitoxin serum level ≥ 33 nmol/l . The specific selection of the analysis and the validation data set is depicted in Fig. The current analyses are based on 901 patients, who had been randomized to digitoxin or placebo in the DIGIT-HF trial prior to March 2021. For the analysis in this manuscript, only patients having an available digitoxin level and a dose adaptation at week 6 could be analyzed. Therefore, patients which have been randomized to placebo or had no available digitoxin level and dose adaptation at week 6 for different reasons  reasons, undetectable digitoxin level due to potential compliance problems) were excluded from this analysis. Finally, 394 patients randomized to digitoxin with available digitoxin levels and dose adaptations at week 6 could be used for the present analysis. A total of 221 patients (56%) maintained a starting dose of 0.07 mg o.d. In 160 patients (41%), the digitoxin dose was reduced from 0.07 mg to 0.05 mg o.d. In 13 (3%) patients, the digitoxin dose was increased from 0.07 mg to 0.1 mg o.d. Patients up-titrated from 0.07 mg to 0.1 mg digitoxin o.d. were excluded, yielding a total of 381 patients suitable for this analysis . These patients were split into a derivation set (Serum samples drawn at study visit 1 (6 weeks after randomization) were sent to a central core laboratory  for determination of digitoxin serum concentrations employing a standardized assay .χ2-test for categorical parameters and two-sided t tests for continuous variables selected from a larger panel of baseline variables of the DIGIT-HF study, based on clinical reasoning and findings reported in the literature. In the second step, we performed an univariable logistic regression analysis including all parameters with a two-sided p value smaller than 5%. For the identification of the most relevant factors, we performed a multivariable logistic regression analysis applying a backward selection procedure with p value criteria for staying in the model of 5%. To identify optimal thresholds for continuous parameters, we performed receiver operating characteristic (ROC) analyses in the next step. Optimal cut-points based on the Youden index were estimated and rounded with respect to clinical meaningful thresholds. Finally, all relevant parameters were equally weighted and the score was built as a sum of the single components. To indentify the score with the highest propability of identifying the patients that correctly received a dose reduction, different combinations of score variables and sum scores were again analyzed using univariable and multivariable logistic regression analyses.First, we identified variables associated with a higher chance of a digitoxin dose reduction. Therefore, we performed univariable two-sided To assess the decisions for the necessity of a dose reduction (yes or no) derived from the dichotomized score, we calculated the sensitivity and specificity in the derivation and validation sets. Sensitivity was defined as the probability that a patient needing dose reduction had been correctly identified, i.e., score ≥ 1. Specificity was defined as the probability that a patient needing no dose reduction had been correctly identified, i.e., score = 0.All analyses were performed with SAS 9.4. Strict blinding regarding individual information on treatment allocation and outcome was ascertained as only one unblinded statistician performing the statistical analyses had access to the data set and all results were displayed to authors only on an aggregated basis.The DIGIT-HF trial is conducted in compliance with the German Drug Law (AMG), the German Good Clinical Practice (GCP) ordinance, ICH GCP guidelines, and other applicable ethical and regulatory requirements. The DIGIT-HF trial is registered at EudraCT (2013-005326-38).n = 317) between the two groups of patients with and without necessity of digitoxin dose reduction identified 6 weeks after randomization. Dose reductions occurred more often in female or older patients, patients with lower body mass index (BMI), lower estimated glomerular filtration rate (eGFR based on the CKD-EPI formula) [First, we descriptively compared baseline variables of the derivation set (formula) , higher 2, eGFR 51 ml/min/1.73m2. As our intention was to develop a simple score, we decided to round up or down to full integers. Since age was kept in the model for clinical reasons, we used the threshold of age ≥ 75 years for the score. The other three components, i.e., female sex, BMI, and eGFR, retained meaningful associations in the multivariable model , or a BMI < 27 kg/m2 . In the final model, the effect for age ≥ 75 years was modest and not statistically significant in the multivariable model: OR 1.22, 95% CI: 0.67–2.22 , but have been described at serum concentrations > 2.0 ng/ml of digoxin and > 30 ng/ml of digitoxin . Based oBecause of their different pharmacokinetic properties, digitoxin represents the pharmacologically more stable cardiac glycoside compared to digoxin, especially in patients with impaired renal function , 7. Lipo, this may result in a lower digitoxin metabolization and potentially higher serum concentrations in women [.In the current analysis, we identified eGFR, BMI, and in particular female sex as factors associated with the necessity of a digitoxin dose reduction in the derivation cohort chosen from the DIGIT-HF population. The strong association with female sex in multivariable regression models might reflect the lower skeletal muscle mass of women compared to men, associated with a reduced compartment and overall digitoxin distribution volume resulting in higher digitoxin serum concentrations in women compared to men for a defined digitoxin dose . Cytochrin women .The next most significant factor associated with the necessity of a digitoxin dose reduction was BMI. BMI, in general, positively correlates with total skeletal muscle mass. Therefore, the observed negative association between BMI and necessity for digitoxin dose reduction reflects, similar as for female sex, the relationship between the compartment and overall volume of digitoxin distribution, which is higher in patients with a higher BMI resulting in the need for higher digitoxin doses to achieve therapeutic serum concentrations.Unexpectedly, we also identified a significant negative association between eGFR and necessity of digitoxin dose reduction. The causal relationship is not clear, because digitoxin elimination is entirely compensated by extrarenal (entero-hepatic) clearance even in patients with impaired renal function and digitoxin serum concentrations are constant even if renal function is markedly impaired . AlthougSpecifically, the correlation between age and eGFR is -0.55 in our data set, which is substantial. Guided by our aim to maximize the probability of detecting all patients who need a dose reduction, we added age to the score and detected in the analysis and the validation set each three more patients for a dose reduction, which increased score sensitivity (data not shown). Therefore, and taking into account knowledge for the need of digitoxin dose reduction because of reduced muscle mass and, therefore, smaller volume of digitoxin distribution in the elderly , age sti2 or a eGFR < 50 ml/min/1.73m2 should start with 0.05 mg digitoxin o.d.. This approach appears easy to implement and may be expected to improve clinical care. Importantly, the digitoxin dosing score was developed based on randomized, placebo-controlled, double-blinded clinical trial data. However, if this score that we propose here will prove useful in clinicial practise will have to be determined, ideally prospectively in a patient population within standard clinical care.The intention of this analysis was to develop a score that faciliatates the dosing decision when treatment with digitoxin needs to be started in HFrEF patients. The components of the score are readily available and with high validity in clinical routine. Female sex, high age, low BMI, and impaired renal function fulfilled these criteria. From the clinical perspective and patient safety, a dosing score should, in particular, prevent overdosing of digitoxin, because digitoxin serum concentrations exceeding the therapeutic range might cause grave side effects or even toxicity. Also, the long half-life of digitoxin leads to prolonged elimination times rendering overdose a significant clinical problem of digitoxin. Therefore, a respective dosing score should have high sensitivity to detect the need of a dose,reduction from 0.07.mg to 0.05 mg o.d. in the present population, which would encourage to start with the low dose of 0.05 mg digitoxin o.d. in clinical practice. Specificity of the score on the other hand is negligible, because we would recommend measurement of digitoxin serum concentration after 6 weeks to check whether a dose of 0.05 mg digitoxin o.d. was sufficient to reach serum concentrations within the therapeutic range. Based on the digitoxin dosing score, all women, and all patients ≥ 75 years or with a BMI < 27 kg/mOur prospective trial data support a retrospective analysis of adverse drug reactions in Germany, indicating adverse drug reactions because of overdosing with digitoxin particular in patients < 70 kg, > 80 years, and women . DespiteBased on recent analyses, cardiac glycosides still may be an important option for the treatment of patients suffering from HFrEF –24. CardA digitoxin dosing score comprising information on age, sex, BMI, and eGFR was derived and validated in patients with HFrEF randomized to digitoxin treatment in the DIGIT-HF trial. The new digitoxin dosing score advises, whether a patient should be started with a low dose of 0.05 mg digitoxin o.d. The digitoxin dosing score can easily be obtained in clinical routine and enables simple and safe initial digitoxin dosing in patients with HFrEF.