The accurate and frequent measurement of the drilling fluid’s rheological properties is essential for proper hydraulic management. It is also important for intelligent drilling, providing drilling fluid data to establish the optimization model of the rate of penetration. Appropriate drilling fluid properties can improve drilling efficiency and prevent accidents. However, the drilling fluid properties are mainly measured in the laboratory. This hinders the real-time optimization of drilling fluid performance and the decision-making process. If the drilling fluid’s properties cannot be detected and the decision-making process does not respond in time, the rate of penetration will slow, potentially causing accidents and serious economic losses. Therefore, it is important to measure the drilling fluid’s properties for drilling engineering in real time. This paper summarizes the real-time measurement methods for rheological properties. The main methods include the following four types: an online rotational Couette viscometer, pipe viscometer, mathematical and physical model or artificial intelligence model based on a Marsh funnel, and acoustic technology. This paper elaborates on the principle, advantages, limitations, and usage of each method. It prospects the real-time measurement of drilling fluid rheological properties and promotes the development of the real-time measurement of drilling rheological properties. In the drilling industry, almost every step requires drilling fluid . The driThe drilling fluid properties play an important role in the optimization of the rate of penetration. In the rate of penetration models established by many scholars, such as mathematical and physical models ,7 and arAlthough the automation of drilling has progressed over the past decade, the current technological control of drilling fluid quality was established 50 years ago and is essentially manual. The American Petroleum Industry’s (API) standard test manual for the petroleum industry requires a complete drilling fluid measurement program to be performed twice a day. At the drilling site, the Marsh funnel is usually used to quickly test the viscosity of the drilling fluid, but this Marsh funnel viscosity can only roughly estimate the viscosity of the drilling fluid, and it cannot reflect all of the drilling fluid’s rheological properties. The rotational Couette standard viscometer can measure all of the drilling fluid’s rheological properties, but they need to be measured in a laboratory after sampling at the site is conducted. If the drilling fluid’s performance does not meet the requirements, it is necessary to test to determine the additive amount to ensure the maintenance of the drilling fluid’s performance. This sample testing and data analysis may take several hours . During The drilling fluid’s rheological properties refer to the characteristics of flow and deformation under the action of external force. The drilling fluid’s rheological properties are essential for the following determinations: estimation of hole cleaning efficiency, calculation of frictional pressure losses in pipes and annuli, determination of equivalent circulating density (ECD) under downhole conditions, determination of prevailing flow regime in pipes and annuli, estimation of swab and surge pressures, and hydraulic optimization for improved drilling efficiency. Proper rheological properties can carry bottom hole cuttings to the ground quickly, increase the rate of penetration, reduce power consumption, ensure drilling safety, and improve economic benefits. In order to estimate the above-mentioned functions of the drilling fluid in time, it is important to measure the drilling fluid rheological properties in real time.τ0 is the fluid yield stress, K is the consistency factor, n is the flow behavior index, and γ is the shear rate. The total ability for a Bingham plastic fluid to resist flow could be expressed by an apparent viscosity (AV) or effective viscosity for a given shear stress [−1 in API.The most common models used to describe the drilling fluid rheology in the petroleum industry are the Bingham plastic model (Equation (1)), Power law (PL) model (Equation 2)), and Herschel–Bulkley model (Equation (3)):), and HeThe Herschel–Bulkley model is used when the accuracy of the rheological parameter measurement is high or in laboratory research. The API recommends using this model; it consistently provides good simulations of measured rheological data for both water-based and non-aqueous drilling fluids. For this reason, it has become the de facto rheological model for engineering calculations in the petroleum industry .The current measurement of rheological properties utilizes a typical manually controlled rotational Couette viscometer. In recent years, many scholars have researched real-time measurement of drilling fluid rheological properties. The real-time measurement technology of drilling fluid rheology mainly includes the following four methods: (1) online rotational Couette viscometer, (2) pipe viscometer, (3) mathematical and physical model or artificial intelligence (AI) model based on a Marsh funnel, and (4) tuning fork technology.−1.The recommended methods for drilling fluid analysis are presented in API 13B . The drin and K), Herschel–Bulkley model , and 10 s and 10 min gel strength can be obtained using a standard rotational Couette viscometer. Many scholars developed an online rotational Couette viscometer based on a standard rotational Couette viscometer to measure the drilling fluid’s rheological properties. The main improvement method is to add a control circuit to control the speed of the motor, turn the reading into an electrical signal output, and add the drilling fluid pipeline to automatically fill the measurement chamber with the drilling fluid. The parameters of the Bingham model  , PL modeSaasen et al. ,22 devel3/h. There were four types of drilling fluid tested, namely, Newtonian fluid, pseudoplastic fluid, water-based drilling fluid, and synthetic-based drilling fluid. The measurement results showed that Newtonian fluid, represented by glycerin, and pseudoplastic fluid, represented by CMC solution, were statically consistent in terms of curve fitting. The results of the water-based mud and non-aqueous drilling fluid found some divergences. Compared with the standard viscometer measurement, the TT-100 tended to underestimate the shear stress in water-based mud, while in the non-aqueous drilling fluid, TT-100 overestimated the shear stress. The limitation of the viscometer was the size of the solids suspended. This article points out that the maximum diameter of the solids in the drilling fluid tested by the instrument must be less than 1 mm. Magalhães et al. [Magalhães et al. ,27 used s et al.  installeDotson et al.  used a dBecause the online rotational Couette viscometer is easily blocked, Vajargah , Magalhãwτ is the wall shear stress (Pa), D is pipe inner diameter (m), ΔP is the friction pressure loss (Pa), and ΔL is the pipe length of the test section (m).In laminar flow, wall shear stress can be obtained when the differential pressure across the measurement section is known:v is the velocity (m/s).It can be shown that for pipes, the shear rate at the wall can be obtained from :(6)γ˙w=1N, as:Introducing the generalized flow behavior index, Then Equation (7) is now rewritten as:wτ vs. ln(8v/D)) represents the generalized flow behavior index, N. Once N is obtained from the flow curve, the shear rate at the wall can be calculated by using Equation (8). Subsequently, rheological parameters for any desired rheological model can be obtained by plotting the shear stress vs. shear rate at the wall and applying a proper curve fitting technique. The Herschel–Bulkley model (Equation (3)) exhibits an acceptable accuracy for the majority of drilling, completion, and cementing fluids and is therefore usually used to fit rheological curves.According to Equation (7), the slope of the “flow curve” (ln ρ is the mud density (kg/m3).Once the rheological constants are obtained, the Reynolds number can be calculated by mud velocity, density, and the wall shear stress as:f is calculated from the frictional pressure loss measurements using:The friction factor Vajargah et al.  designedR2) was 0.99. In experimental verifications, a total of fifteen tests were performed using various water-based mud and oil-based mud formulations at 25 °C, 50 °C, and 65.5 °C. It showed the precision and robustness of the pipe viscometer method and that it could be used to provide a quality and frequent fluid characterization for field muds. In field testing, the pipe viscometer measurements of both PV and YP were a perfect match to the data reported in daily mud reports by the mud engineer.p is the pressure loss (Pa), i is each measured data point, and n is the total number of measurements.The pipe viscometer designed by Sercan Gul et al.  has the Taugbøl et al. ,34 use tMagalhães Filho et al.  comparedSome methods have the same theory as the pipe viscometer. Compared with the abovementioned pipe viscometers, these methods are convenient and do not take up much space. A novel downhole sensor was developed by Rondon et al. . It can 3 was prepared, and 0.1% polyacrylamide glue solution, 0.1% polyacrylonitrile ammonium, and xanthan gum was added successively. The pipe viscometer continuously recorded the change process of the rheological properties of the drilling fluid, the measurement results were the same as the standard viscometer measurement results, and the error was small. Sun et al. [Pipe viscometers usually use a round pipe, the sensor installed in the round pipe may affect the flow rate, resulting in inaccurate pressure measurement. Therefore, Liu et al.  developen et al.  develope−3, and the mean absolute percentage error was 4.55%. The algorithm was developed using the trained machine learning model and the pipe viscometer equations. The rheogram results matched the standard Couette viscometer.While a pipe viscometer takes up a lot of space, Sercan Gul ,46 develt is March funnel time, seconds.Marsh funnel is a commonly used instrument for viscosity analysis, calculating the final fluid release time of almost 1.5 L. Marsh  inventedAn artificial neural network (ANN) is an effective technology that imitates the biological neurons of the human brain . The maiK, n, and τ0).According to the above theories, artificial intelligence methods are used to estimate rheological properties more accurately in real time based on parameters such as the Marsh funnel viscosity, mud weight, and solid content ,69,70,71Abdelgawad et al.  and Elkan, K, τ0, reading of 300 rpm and 600 rpm) in real time and show good results. The methods to evaluate the performance of the ANN model are average absolute error (AAE) (Equation (16)), average absolute percentage error, AAPE (Equation (17)), R2, and mean square error (MSE). In all these papers, the predicted result for R2 was greater than 0.89, AAE was less than 4.7, and AAPE was less than 8.6%. This inexpensive technique will help drilling engineers to control the drilling operation better and predict drilling problems before they occur. Moreover, it will reduce the total cost of drilling operations. However, different ANN models need to be established by training drilling fluid data in different mud systems. One artificial intelligence model can be used in wells located in the same block or in the same drilling fluid system.i is each measured data point, realy is real measured value, and predicty is predict measured value.As shown in In 2011, Miller et al.  introducThe tuning fork instrument kit operates at a back pressure of 0.7–0.8 MPa, so the density read is equal to the density of the normal pressurized drilling fluid. Density and viscosity are calibrated by manual readings per shift. The instrument kit can be recalibrated based on the new readings of the standard Marsh funnel viscometer when the density or viscosity significantly changes. Experience in fields showed that a calibration check is sufficient at the start of each shift to ensure that readings are comparable directly to manual readings. The sensor is small and easy to install but requires manual calibration.Ofoche et al.  presents(1)The online Couette viscometer is the most similar to the API standard measurement method, so it has the highest accuracy and can measure all drilling fluid rheological properties. However, the gap between the rotor and the stator is narrow, and the diameter of solids must less than 1 mm. Solid or gels particles may be sedimented in the viscometer, so the online Couette viscometer is easily plugged. It is inconvenient to use and requires regular cleaning and maintenance. This viscometer is suitable for drilling fluids with low viscosity and low solid content.(2)Compared with the online Couette viscometer, the pipe viscometer provides better automatic measurement technology. The solid and gel particles in the drilling fluid will not settle in the pipe. By adding additional sensors to the pipe, additional variables can be obtained such as fluid density, temperature, critical Reynolds number, and real-time friction coefficient. However, it cannot measure the 10 s and 10 min gel strength. The pipe viscometer is large, and it occupies a large space for installation. Compared with the pipe viscometer, the helical pipe viscometer has obvious advantages, having a compact size and more general friction pressure loss curve. At the same time, the helical pipe increases the friction pressure loss and delayed flow state transition, so the helical pipe viscometer can be used to collect more data in the laminar flow state, thereby improving the accuracy of low shear rheological parameter estimation. However, the theoretical basis for the helical pipe viscometer is still under development.(3)Artificial intelligence technology is the cheapest method, because only the Marsh funnel is needed, and the mud balance and solid content meter may be added optionally. Although the test of the Marsh funnel, density, and sand content was simple and quick, it still required manual testing. The neural network model was different when the drilling system was different. One artificial intelligence method can be used in wells which are in the same block or in the same drilling system. The test results of the tuning fork technology were Marsh funnel viscosity and density, which can be combined with artificial intelligence technology to form an automatic online measurement of drilling fluid rheological properties.(4)The current drilling fluid rheology measurement is offline in API recommended practice for field testing drilling fluids, which can no longer meet the needs of intelligent drilling. In order to control and optimize rheological parameters in real-time, it is necessary to consider developing a criterion that can measure drilling rheological properties in real time. The standard Couette viscometer also has shortcomings ,76,77,78(5)The practicability of the instrument is relatively not good. We think two convenient methods can be considered in the future. One method is using MWD technology to measure the pressure in the drill string and annulus while drilling, the ECD can be accurately obtained. The drill pipe and annulus are considered as a large pipe viscometer, which can calculate the drilling fluid rheological properties. The other method is acoustic technology, including tuning fork technology, ultrasonic technology, etc. There are many articles using ultrasound to measure fluid parameters ,81,82,83(6)The current drilling fluid performance testing mainly measures the drilling fluid that samples from the mud pit. The drilling fluid returning from the annulus contains a lot of stratum information, so its testing is also very important and can help judge the formation. Testing its performance can also make better decisions for processing to maintain the performance of drilling fluid. However, the mud returning from the annulus contains many solid particles, and some particles have large diameters. Therefore, the allowable particle diameter of the instrument needs to be further considered in the selection of equipment.This article analyzes the four real-time measurement technologies of drilling fluids, and the measurement results of each technology are within the tolerance range compared with the standard rotational Couette viscometer in field test. At present, there is no industry standard handbook for real-time drilling fluid measurement. This status hinders the automatic control of drilling fluid. In the future, technical standards for real-time drilling fluid measurement will be established based on standard laboratory testing methods. This article analyzed the four methods in terms of principles, implementation methods, advantages, limitations, etc. Engineers can choose one of the four methods for real-time measurement according to their requirements in the field. The online rotational Couette viscometer is suitable for the low-viscosity and low-solid content drilling fluid. The pipe viscometer is a reliable real-time measurement method. As a result, a standard and small pipe viscometer may be formed to test the drilling fluid rheological properties in the future. The technology of MWD downhole pressure measurement and ultrasound technology can also be considered to measure drilling fluid rheological properties. Through real-time measurement of the rheological properties of drilling fluids, the performance of drilling fluids can be grasped in real-time, the drilling status can be judged in time, and relevant drilling fluid processing decisions can be made to ensure drilling safety. Through big data, artificial intelligence, and other technologies, the drilling fluid performance can be optimized in real time to achieve the optimal rate of penetration, thereby improving economic benefits.


To the Editor,In their hypothesis paper, Wirth, Scheibenbogen, and Paul describe how endothelial dysfunction could produce a wide range of neurological symptoms in people with ME/CFS . As theyFor the past four years, my wife and I have been caregivers for our teenage daughter, who has ME/CFS, hypermobile Ehlers-Danlos syndrome, craniocervical instability, Chiari malformation and several other comorbid conditions. Through observation and trial and error, I have developed a number of hypotheses on these matters that I offer here in the hope they might prompt formal research into how to effectively treat these conditions .Discussion of endothelial dysfunction and vascular damage in ME/CFS and Long COVID generally focuses on how leakages from dysfunctional blood vessels lead to reduced blood flow, which has many consequences, including reduced oxygenation of muscles and reduced cerebral brain flow. As researchers study this phenomenon, I would encourage consideration of the additional possibility that the leaking fluid causes independent damage. Lipedema researchers have found that leakages from microangiopathic blood vessels cause an excess of interstitial fluid that stimulates the formation of subcutaneous adipose tissue , which gI hypothesize that a similar process happens when fluid leaks from faulty blood vessels in ME/CFS, possibly exacerbated by endothelial dysfunction in lymphatic vessels that inhibit the fluid’s removal, causing widespread pain and inflammation. This mechanism appears most pronounced among people with hypermobility or other connective tissue disorders, a common trait among people with both ME/CFS and lipedema.My daughter experiences pain from fibrotic adipose tissue as well as what appears to be nerve compression from accumulated interstitial / lymphatic fluid. Manual lymphatic drainage, the squeezing of affected tissue, and the manual break-up of fibrotic adipose tissue have helped to ameliorate these symptoms.In my daughter, I have also observed impaired drainage of fluid from the glymphatic system, both at the cribriform plate and down her spine. Could this be related to damaged lymphatic vessels or blockages from fibrotic adipose tissue?Like many people with moderate or severe ME/CFS, my daughter struggles to recover from even small amounts of physical exertion. In addition to mitigating her pain, manual lymphatic drainage and the squeezing of affected tissue greatly accelerates this recovery process. We have observed a direct dose–response relationship: the more exercise, the more fluid is present in her tissues, and the more manual draining / squeezing is necessary for her to recover.Based on this experience, I hypothesize that excess interstitial fluid resulting from dysfunctional blood and lymphatic vessels contributes to the experience of post-exertional malaise, with fluid literally drowning affected tissue, leading to hypoxic conditions and inflammation. Possible explanations for the increased interstitial fluid are increases in blood pressure during physical exertion, hypermobile joints going out of place, prompting localized increases in interstitial fluid, and increases in cortisol that generate an increase in fluid and blood volume. Increases in fluid leakage due to elevated cortisol levels may also explain why some people with ME/CFS feel worse when stressed or anxious. The role of cortisol (or another mediator with fluid retaining properties) may explain why cognitive exertion can also generate post-exertional malaise. When present, elevated estrogen levels may exacerbate leakage by increasing fluid volume.I am not sure why there is typically a delay between physical exertion and the experience of the most acute symptoms of post-exertional malaise. One possibility is that it takes time for the tissue inundated with fluid to feel the full effects of the hypoxic conditions. Another possibility is that a biphasic reaction triggered during physical exertion leads to the release of a mediator that causes heightened endothelial dysfunction and fluid release.Further research is needed into the causes of endothelial dysfunction and damage  and appropriate treatment.


Enterobacterales (CREs). Carbapenemase-negative (CN) CRE isolates have been reported, but the activity of newer agents against these isolates is still not well understood. We evaluated the activity of MVB against CN-CRE collected during 6 years of surveillance in US hospitals.Meropenem-vaborbactam (MVB) is an important addition to the armamentarium to treat infections caused by carbapenem-resistant Enterobacterales isolates collected in US hospitals from 2014-2019 were susceptibility tested by reference broth microdilution methods. Results were interpreted using CLSI 2020 breakpoints. CRE isolates were submitted to PCR/sequencing (2014-2015) or whole genome sequencing  for characterization of carbapenemase genes. Isolates from 2016-2019 were evaluated for other beta-lactam resistance mechanisms.A total of 27,968 K. pneumoniae (16 isolates) followed by E. cloacae (9), E. coli (8), and K. aerogenes (8). MVB was the most active agent tested against these isolates, inhibiting 47/48 (97.9%) of the isolates tested. The only isolate displaying a resistant MIC for MVB was a P. mirabilis  collected in 2015. Meropenem alone inhibited only 2.1% of the isolates. Other beta-lactams inhibited 4.2 to 14.6% of the isolates. Among non-beta-lactam comparator agents, tigecycline and amikacin inhibited 93.8 and 91.7% of the isolates, respectively, when applying CLSI or US FDA breakpoints. A total of 89.6% of the isolates had intermediate colistin MIC values. Among the 27 isolates collected from 2016-2019 that were submitted to WGS, 15 harbored CTX-M encoding genes. K. aerogenes and E. cloacae isolates (3 each) overexpressed AmpC. OmpC/K36 was disrupted in 20 isolates and OmpF/K35 was disrupted in 8 isolates.Among 357  CRE isolates identified during 6 years of surveillance, 48 (13.4% of the CRE) isolates did not produce known carbapenemases. The CN-CRE collection included 7 bacterial, species, or species complex. The top four most common species in the collection were MVB displayed good activity against CN-CRE isolates from US hospitals. This combination agent could be a good option to treat infections caused by these isolates.Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  Involved: Self): Research Grant or Support; BioVersys Ag  Involved: Self): Research Grant or Support; Bugworks  Involved: Self): Research Grant or Support; Cidara  Involved: Self): Research Grant or Support; Cipla  Involved: Self): Research Grant or Support; Contrafect  Involved: Self): Research Grant or Support; Cormedix  Involved: Self): Research Grant or Support; Crestone, Inc.  Involved: Self): Research Grant or Support; Curza  Involved: Self): Research Grant or Support; CXC7  Involved: Self): Research Grant or Support; Entasis  Involved: Self): Research Grant or Support; Fedora Pharmaceutical  Involved: Self): Research Grant or Support; Fimbrion Therapeutics  Involved: Self): Research Grant or Support; Fox Chase  Involved: Self): Research Grant or Support; GlaxoSmithKline  Involved: Self): Research Grant or Support; Guardian Therapeutics  Involved: Self): Research Grant or Support; Hardy Diagnostics  Involved: Self): Research Grant or Support; IHMA  Involved: Self): Research Grant or Support; Janssen Research & Development  Involved: Self): Research Grant or Support; Johnson & Johnson  Involved: Self): Research Grant or Support; Kaleido Biosceinces  Involved: Self): Research Grant or Support; KBP Biosciences  Involved: Self): Research Grant or Support; Luminex  Involved: Self): Research Grant or Support; Matrivax  Involved: Self): Research Grant or Support; Mayo Clinic  Involved: Self): Research Grant or Support; Medpace  Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd.  Involved: Self): Research Grant or Support; Melinta  Involved: Self): Research Grant or Support; Menarini  Involved: Self): Research Grant or Support; Merck  Involved: Self): Research Grant or Support; Meridian Bioscience Inc.  Involved: Self): Research Grant or Support; Micromyx  Involved: Self): Research Grant or Support; MicuRx  Involved: Self): Research Grant or Support; N8 Medical  Involved: Self): Research Grant or Support; Nabriva  Involved: Self): Research Grant or Support; National Institutes of Health  Involved: Self): Research Grant or Support; National University of Singapore  Involved: Self): Research Grant or Support; North Bristol NHS Trust  Involved: Self): Research Grant or Support; Novome Biotechnologies  Involved: Self): Research Grant or Support; Paratek  Involved: Self): Research Grant or Support; Pfizer  Involved: Self): Research Grant or Support; Prokaryotics Inc.  Involved: Self): Research Grant or Support; QPEX Biopharma  Involved: Self): Research Grant or Support; Rhode Island Hospital  Involved: Self): Research Grant or Support; RIHML  Involved: Self): Research Grant or Support; Roche  Involved: Self): Research Grant or Support; Roivant  Involved: Self): Research Grant or Support; Salvat  Involved: Self): Research Grant or Support; Scynexis  Involved: Self): Research Grant or Support; SeLux Diagnostics  Involved: Self): Research Grant or Support; Shionogi  Involved: Self): Research Grant or Support; Specific Diagnostics  Involved: Self): Research Grant or Support; Spero  Involved: Self): Research Grant or Support; SuperTrans Medical LT  Involved: Self): Research Grant or Support; T2 Biosystems  Involved: Self): Research Grant or Support; The University of Queensland  Involved: Self): Research Grant or Support; Thermo Fisher Scientific  Involved: Self): Research Grant or Support; Tufts Medical Center  Involved: Self): Research Grant or Support; Universite de Sherbrooke  Involved: Self): Research Grant or Support; University of Iowa  Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics  Involved: Self): Research Grant or Support; University of Wisconsin  Involved: Self): Research Grant or Support; UNT System College of Pharmacy  Involved: Self): Research Grant or Support; URMC  Involved: Self): Research Grant or Support; UT Southwestern  Involved: Self): Research Grant or Support; VenatoRx  Involved: Self): Research Grant or Support; Viosera Therapeutics  Involved: Self): Research Grant or Support; Wayne State University  Involved: Self): Research Grant or Support Timothy B. Doyle, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Valerie Kantro, n/a, Melinta Therapeutics, Inc. (Research Grant or Support) Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Dee Shortridge, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support)


We have found that the apparent polarity of the organic phase is only modulated when DNNS− binds to large polyions such as protamine, unlike singly charged ions that lack the cooperative binding required to cause a significant shift in the distribution of the polarizing DNNS− ions. Our new sensing mechanism allows solvatochromic signal transduction without the transducer undergoing ion exchange. The result is significantly improved sensitivity and selectivity, enabling for the first time the quantification of protamine and heparin in human plasma using optical nanosensors that correlates with the current gold standard analysis method, the anti-Xa factor assay.Optical nanosensors for the detection of polyions, including protamine and heparin, have to date relied upon ion-exchange reactions involving an analyte and an optical transducer. Unfortunately, due to the limited selectivity of the available ionophores for polyions, this mechanism has suffered from severe interference in complex sample matrices. To date no optical polyion nanosensors have demonstrated acceptable performance in serum, plasma or blood. Herein we describe a new type of nanosensor based on our discovery of a “hyper-polarizing lipophilic phase” in which dinonylnaphthalenesulfonate (DNNS Novel optical nanosensors for the selective detection of the polycationic protamine based on solvatochromic signal change allow one to detect heparin in plasma. A significant amount of research has since been carried out to ascertain whether polyions adhere to the electrode membrane surface or are extracted into the membrane bulk. Early in the development of polyion-selective electrodes, a response theory was proposed based solely on bulk extraction principles, which was found to describe the experimental behavior adequately, indicating that the bulk extraction of polyions may be the dominant mechanism.3 However, optical microscopy carried out by Meyerhoff and coworkers suggested significant surface accumulation of protamine.8 This was followed by ion transfer voltammetric measurements by Amemiya and coworkers concluding that both adhesion and bulk extraction processes take place in parallel,9–11 later confirmed by the group of Meyerhoff.12Ion-selective electrodes (ISEs) are among the most robust sensors for the analysis of electrolytes in blood.13–15 Early examples of optical protamine and heparin sensors used cast sensing films and showed promising performance in diluted serum.16,17 Following this work, membrane and particle type sensors that rely on an ion exchange between the target ion and an optical reporter were developed further.18–22 Emulsified (nanoparticle-type) optical sensors have been of particular interest as they allow assay miniaturization , integration of automated liquid handling, and significantly reduced equilibrium times. Unfortunately, nanoparticle-based polyion sensors for reliable measurement in blood, plasma and serum have not yet been successful. For example, Xie and coworkers have reported that the signal of protamine sensors containing dinonylnaphthalenesulfonate (DNNS−), a commonly used protamine receptor, was found to completely saturate upon addition of serum to sensors, pointing to important matrix effects25 and the need for extreme dilution of serum samples.26Following the developments with ISEs, ion-selective optical sensors have emerged as powerful variants of ISEs that allow for an optical readout of ionic species, including polyions.27In this work protamine and heparin were chosen as model target polyions as convention, and we describe protamine-selective nanoscale optodes employing a fundamentally novel sensing mechanism for assaying heparin in human plasma samples by protamine titration. The research first explores an ion exchange-based assay involving capillary coated sensing films, the basis of which was previously described for the detection of potassium in human serum.− : DOS at a 2.8 : 1 weight ratio, thereby DNNS− acts as a solvent rather than a solute. The electron acceptor moiety of solvatochromic dyes strongly interacts with the sulfonate group of DNNS−, producing a stronger bathochromic shift than even that of an aqueous environment in the organic phase.Following this work, we will describe our finding of a “hyper-polarizing organic phase” that forms the basis of a new sensing mechanism. This hyper-polarizing organic phase was produced by adding a large excess of DNNS−/dye interaction resulting in a large hypsochromic shift, which fascinatingly was not induced at all by singly charged species including arginine, the main repeating amino acid moiety of protamine. This charge/size cutoff resulted in significantly better selectivity than any previously reported optical nanosensor for polyions relying on ion exchange of sensing components. This feature of our new nanosensor is likely due to the differences in cooperative binding between large and small molecules, which will be discussed in more detail in the Discussion.Upon creating nanosensors with a hyper-polarizing organic phase, we have found that only relatively large molecules or proteins such as protamine can disrupt the DNNS+ and SD017. The improved performance allowed both sensors to quantify protamine in concentrated serum. Due to superior optical properties, the X3-based sensor was then taken forward for quantifying protamine in plasma for the first time using an optical nanosensor. The success of protamine quantification in plasma ultimately allowed for the detection of heparin in plasma samples with the X3+-based nanoparticle sensor by protamine titration. This was achieved in the narrow concentration range of 0–0.8 U mL−1, which is of high demand for heparin level monitoring during heparin neutralization with protamine as a protamine overdose poses a lethal risk and for the treatment of thrombosis patients with heparin which should be kept at around 0.5 U mL−1 in blood. The performance was evaluated with clinical samples and cross-correlated with the anti-Xa assay, the gold standard routinely used in hospitals.Hence, this nanosensor based on a hyper-polarizing organic phase has two fundamental advantages over previously reported sensors: sensitivity due to the larger solvatochromic shift and selectivity due to the size-filtering effect. This trend was seen with two different nanoparticle formulations containing the solvatochromic dyes X327 An analogous system for the measurement of protamine was explored, using the positively charged dye Malachite Green (MG+) as the counter ion of the protamine recognition unit dinonylnaphthalene sulfonate, DNNS−. Upon exposure to a protamine-containing sample, protamine is exchanged for MG+  using either DNNS+, was explored which exhibits a greater solvatochromic absorbance shift. This dye is also more hydrophilic. + in water (dark blue trace), showing a peak maximum around 505 nm. In an organic solvent (DOS) containing a lipophilic, non-coordinating cation-exchanger, TFPB−, the absorbance peak red shifts to about 545 nm. The four sterically bulky phenylborate groups of TFPB− are known to symmetrically distribute the negative charge.23 If this bulky ion-exchanger is replaced by DNNS−, used to recognize protamine, the absorbance is found to blue shift to a maximum around 495 nm . A molar ratio of DNNS− : DOS of 1 : 1 was used here, in contrast to 2.8 : 1 in the actual nanosensor, because of the high absorbance of DNNS− which placed spectrophotometric limits on this experiment. For this reason a larger blue shift (peak at 460–480 nm) was observed for the emulsified nanosensor particles.Because the dye SD017 gave relatively small spectral changes, another solvatochromic dye, X35 nm see . This is−, disrupting the association with X3+ and hence resulting in a shift of the peak maximum to longer wavelengths. The nanoparticle sensor for protamine detection contained the solvatochromic dye X3+ as an optical transducer along with the lipophilic cation-exchanger TFPB− as a counter ion to stabilize it in the emulsified organic sensing phase. The combination of just these two reagents did not result in a response to protamine, see −, increasing protamine levels continuously decreased the initial peak at 480 nm, forming a new red shifted peak around 545 nm. This suggests that the initial interaction between the solvatochromic dye X3+ and DNNS− is continuously disrupted by increasing levels of protamine. When their interaction is completely disrupted, the spectrum of the nanosensor (see cprotamine) looks very similar to that of the combination of X3+ and TFPB− ). The main difference between the two mechanisms is where protamine settles. Both are in principle possible because protamine and other polyions have been reported to extract to the organic sensing phase and also to be adsorbed at the interface, as evidenced by electrochemical and optical experiments (see the Introduction for more details). The observed zeta potential changes in the absence and presence of protamine are shown in Table S1.Based on this, two different mechanisms are proposed see  right. I− concentration in the sensing phase, which was here experimentally changed through the plasticizer concentration. Fig. S4The new sensing mode is found to depend strongly on the DNNS− (yellow triangle). The response of the sensor with a lower DNNS− concentration is clearly linear in nature, indicating exhaustive operation. Interestingly, the response for the high DNNS− concentration becomes weaker at low protamine concentration. This may be caused by the excessive DNNS− already decorating the solution–particle interface in the absence of protamine, which will no longer modulate inner particle polarity when protamine starts being present. Although DNNS−/protamine complexation is exhaustive, the signal transduction range can be tuned in this manner. Because the sensing mechanism is exhaustive,20 the sensitivity of the system can be further modulated by altering the total particle concentration.The sensor operates in an exhaustive manner in which nearly all protamine molecules in the sample solution are extracted into (or onto) the sensor nanoparticles. Fig. S5A selectivity test using arginine (Arg), the main repeating amino acid of protamine, suggests that the nanosensors developed here function in a different manner to conventional sensors. As shown in 2+, Mg2+ and Na+  sourced from the University Hospital of Geneva (HUG) for which cross-correlation data with anti-Xa assay were available. These heparin levels are significantly lower and analytically more challenging than the range reported earlier with potentiometric heparin sensors (1–10 U mL−1heparin). Consistent with expectations, the nanosensors were confirmed to respond independently of pH in a wide range (pH 5–9), see Fig. S9,− sulfonate group, but this would be outside the physiological range.The nanosensors were used to quantify low heparin concentrations in plasma samples . We assume that the error observed here arose from the matrix effect that differs from patient to patient. Our new sensors are also capable of heparin level analysis at higher concentration relevant to open heart surgery as demonstrated in Considering the previously reported cross-correlations of various newly developed sensors and devices specifically towards the anti-Xa assay,l-lysine (polylysine) can also be measured using our new sensor as shown in Fig. S15.+), a cationic ligand used to induce polarisation. Based on the above results, we believe that the described hyperpolarization-based sensor possesses broad potential for the measurement of other polyions and will facilitate nanoscale polyion sensing in biological samples.As shown above, the new sensing mechanism has enabled protamine and heparin analysis in concentrated serum and plasma samples. Moreover, the application of our sensor is not limited to only these two polyionic species. For example, α-poly-−, a receptor for protamine, dramatically alters the internal particle polarity in the absence and presence of protamine, allowing for a solvatochromic signal transduction. Although the detailed mechanism is not yet entirely clear, experimental data suggested two possible mechanisms involving either the extraction of protamine into the sensing phase or its binding to the sensing particle surface. The new nanosensor showed an excellent improvement in selectivity compared to conventional ion exchange type sensors, which allowed protamine quantification in plasma and serum. Notably, the new sensor showed complete absence of interference from arginine, the major amino acid component of protamine, which is in contrast to conventional sensors. The promising performance of the new sensor strategy was confirmed in 17 patient samples by comparing it to the anti-Xa assay used as the gold standard. We are now developing new device strategies based on this chemistry to further reduce biological fouling and enhance heparin quantification capabilities.We have developed a new protamine/heparin sensor in which DNNSin situ for optical sensing of polyion polymerization processes due to the size effect in which hypsochromic effects are observed as arginine polymerization proceeds.We believe that our sensing mechanism can be applied to other combinations of receptors and polyions. In our laboratory, nanosensors for biologically relevant polyanions such as DNA, RNA and phosphorylated proteins are under development. In addition, this nanosensor should in principle be able to operate 34 This approach would exclude blood cells from the gel-based sensing phase and allow for the determination of protamine/heparin in whole blood.The application of these newly developed sensors to whole blood may be achieved by embedding these sensors in a gel, in analogy to the recent work by Xie's group.Conceptualization, Y. S. and E. B.; experiments, Y. S., R. N. and K. J. R.; writing – original draft, Y. S. and K. J. R.; writing – review and editing, Y. S., K. J. R. and E. B.; supervision, E. B.There are no conflicts to declare.SC-012-D1SC04930E-s001


Periosteal chondrosarcoma is an extremely rare malignant cartilage-forming tumour that originates from the periosteum and occurs on the surface of bone. Often, it is difficult to distinguish periosteal chondrosarcoma from other tumours, and reports in the literature are scarce. This study aims to investigate the characteristics of periosteal chondrosarcoma, focusing particularly on medullary invasion. Among 33 periosteal cartilaginous tumours, seven patients with pathologically proven periosteal chondrosarcoma were identified retrospectively. The average tumour size was 5.4 cm in the long axis; two tumours were smaller than 3.0 cm. Six tumours were resected with a wide margin, and the remaining tumour had a marginal margin. Histology revealed that six tumours (85.7%) had invaded the medullary cavity; three of these did not show invasion into the medullary cavity on MRI evaluation. Neither local recurrence nor metastasis was observed among these patients. The frequency of invasion of the medullary cavity was higher than that reported previously. The recommended treatment for periosteal chondrosarcoma is resection with an adequate margin. Therefore, surgeons should consider the possibility of medullary invasion when attempting to achieve a histologically negative margin, even if the tumour does not show invasion into the medullary cavity on MRI. Periosteal chondrosarcoma is a malignant cartilage-forming tumour that originates from the periosteum and grows on the surface of the bone . The tumPeriosteal chondrosarcoma arises from the periosteum and, usually, grows on the outside of the bone; however, it can occasionally extend into the medullary cavity ,6,7,8. AIn this case study, we investigate the clinical and histological characteristics, treatments and clinical outcomes of seven patients with periosteal chondrosarcoma, focusing particularly on the risk of medullary invasion.Thirty-three patients diagnosed with a periosteal cartilaginous tumour were retrieved from the bone and soft tissue tumour archives 1998–2020) of the three participating institutions. The records were reviewed retrospectively and seven patients with pathologically proven periosteal chondrosarcoma were identified. The following clinical data were extracted from the records: (1) demographics and preoperative variables , tumour size and radiological features); (2) therapeutic variables ; and (3) oncologic outcome . Symptoms (presence or absence of pain or swelling) at the time of presentation were recorded, as was symptom duration . Radiological features were assessed using conventional radiographs, computed tomography (CT) and magnetic resonance imaging (MRI). Conventional radiographs and CT evaluation included an assessment of tumour shape, lesion mineralisation and cortical erosion or thickening. Tumour size was measured on preoperative MRI, and intrinsic signal characteristics, soft tissue oedema and medullary invasion were analysed. All cases were reviewed and confirmed by two experienced bone tumour surgeons and pathologists. Although histological grading of periosteal chondrosarcoma is not part of the 2020 World Health Organisation classification, it was performed in accordance with the methods used for conventional chondrosarcoma [998–2020 Pathological review revealed that seven of the 33 patients had been diagnosed with periosteal chondrosarcoma, whereas the remaining 26 were diagnosed as periosteal chondroma. Patient characteristics are summarised in n = 3, 42.9%), followed by the metaphysis of the proximal humerus  (n = 2), cortical thickening (n = 3) and a periosteal shell (n = 7). For all tumours, T1-weighted MRI showed homogeneous low signal intensity masses, whereas T2-weighted MRI showed predominantly high signal intensity masses. In three cases (42.9%), medullary invasion was observed on T2-weighted MRI (n = 4).All patients underwent conventional radiography and MRI; six tumours were also assessed by CT. The metaphysis of the distal femur was the most common tumour site  . The mea, 28.6%) a,b. Othehted MRI c,d. OtheOf the five cases that underwent open biopsy, three were diagnosed as periosteal chondrosarcoma and all were resected with a wide margin. Of the two cases that did not undergo open biopsy, one (Case 1) was resected with a wide margin because cortical erosion and soft tissue oedema were observed, and malignancy was clinically suspected. In the other case (Case 4), preoperative imaging made it difficult to distinguish benign from malignant. Therefore, the fibula and the extra-skeletal lesion were resected en bloc and with a marginal margin, respectively. To reconstruct the bone defect after tumour resection, two patients received endoprostheses, two received an autogenous bone graft and one received beta-tricalcium phosphate (β-TCP). Two patients did not require reconstruction of the bone defect because they had a tumour in the fibular diaphysis and pubis, respectively. Histologically, all tumours showed lobular proliferation of atypical chondrocytes with enlarged nuclei; double-nucleated cells and moderately increased cellularity were also observed. Three patients were diagnosed with periosteal chondrosarcoma grade I (42.9%) and the other four were diagnosed with grade II (57.1%). A cortical bone-permeating pattern was seen in all tumours; however, this was not observed in 26 periosteal chondromas . FurtherHere, we reviewed seven patients with periosteal chondrosarcoma and present the clinical and pathological characteristics, treatments and outcomes. Of note, careful pathological evaluation revealed that the rate of medullary invasion in this cohort  was higher than that reported previously [Generally, chondrosarcoma is resistant to chemotherapy and radiation therapy; therefore, surgery is the first treatment of choice. Resection with an adequate margin is strongly recommended for periosteal chondrosarcoma because reports suggest that inadequate excision results in local recurrence and pulmonary metastasis ,3,9. At The main differential diagnoses for periosteal chondrosarcomas are periosteal chondroma, periosteal osteosarcoma, or secondary peripheral chondrosarcoma. Periosteal chondroma is the most difficult tumour to differentiate from periosteal chondrosarcoma because the two have many overlapping features on imaging. Periosteal chondroma causes no cortical destruction, whereas periosteal chondrosarcoma often shows cortical destruction , althougThe study has several limitations. First, data from patients with periosteal chondrosarcoma were collected from three different hospitals. There were potential differences in diagnostic methods and treatment philosophies, although all the patients were treated by expert surgeons specialised in sarcoma. Also, all data were reviewed by the first and the corresponding authors. Second, the relatively small number of patients limits the power of the study. We performed a multi-institutional study because periosteal chondrosarcoma is extremely rare. We believe that this study provides useful information about tumour characteristics as it is one of very few to examine this rare tumour in detail.In summary, we present a rare cases series comprising seven patients with periosteal chondrosarcoma in which we evaluated clinical and pathological characteristics, treatments and outcomes. The rate of medullary cavity invasion was higher than that reported previously. Therefore, a wide resection is recommended, and surgeons should pay close attention to the possibility of medullary invasion, even if the tumour does not invade the medullary cavity on MRI.We evaluated medullary invasion of periosteal chondrosarcoma both radiologically and histologically and found that the frequency was higher than that reported previously. Therefore, surgeons should consider the possibility of medullary invasion at surgery planning in order to achieve a histologically negative margin, even if the tumour does not show invasion into the medullary cavity on MRI.


The molecular and cellular mechanisms underlying mammary tumour dormancy and cancer recurrence are unclear and remain to be elucidated. Here, we report that mammary epithelial-specific disruption of β1 integrin in a murine model of Luminal B human breast cancer drastically impairs tumour growth with proliferation block, apoptosis induction and cellular senescence. β1 integrin-deficient dormant lesions show activation of the tumour suppressor p53, and tumours that circumvent dormancy possess p53 mutation analogous to those in human disease. We further demonstrate that mammary epithelial deletion of p53 in β1 integrin-deficient mice fully rescues tumour dormancy and bypasses cellular senescence. Additionally, recurrent β1 integrin-deficient tumours exhibit fibrosis with increased cancer-associated fibroblast infiltration and extracellular matrix deposition, absent in fast-growing β1 integrin/p53-deficient lesions. Taken together, these observations argue that β1 integrin modulates p53-dependent cellular senescence resulting in tumour dormancy and that pro-tumourigenic stromal cues and intrinsic genetic mutation are required for dormancy exit. Tumour dormancy is a major hurdle in the clinical management of breast cancer due to its detrimental roles in therapy resistance and lethal disease recurrence. Dormancy can occur at both cellular and tumour levels, referred to as cellular dormancy and tumour mass dormancy, respectively . DormantEmerging studies of breast cancer have demonstrated the importance of the extracellular matrix (ECM) and integrin receptors not only in promoting breast malignancy but also in regulating the maintenance of and the exit from dormancy , 4. InteItgb1). Consistent with our previous results, mammary-specific ablation of β1 integrin significantly inhibited tumour initiation and progression to invasive disease, resulting in the formation of dormant lesions with slow tumour growth rate correlated with increased cell cycle arrest, apoptosis and cellular senescence. We further showed activation of tumour suppressor p53 in dormant tumours, while p53 inactivation alleviated dormant cell behaviours and enabled the exit of tumour mass dormancy. Additionally, dormant tumours were found to evolve within a permissive microenvironment associated with aberrant fibrosis and infiltration of activated matrix cancer-associated fibroblasts. Taken together, the results indicate that our β1 integrin-deficient GEMM model recapitulates all stages of tumour mass dormancy, including induction, maintenance, and re-emergence, and that re-emergence from dormancy involves cell intrinsic and extrinsic alterations which facilitate both cancer cell survival and proliferation.However, one key caveat of this β1 integrin-deficient transgenic system is that the stochastic expression of MMTV/Cre transgene in the PyV mT model gave rise to ‘escapee’ tumours retaining β1 integrin expression due to the lack of Cre recombinase expression . To circOne major gap in our understanding of tumour dormancy is the lack of appropriate immune competent model that recapitulates the different stages of tumour dormancy. To address this issue, we generated the MIC GEMM that has several advantages over the conventional MMTV-PyV mT model Fig.  11]. In. In11]. To further evaluate the effects of β1 integrin ablation on early stages of mammary tumourigenesis, histopathological analyses of MIN structures at 2 week-post Dox induction revealed solid adenoma lesions in wild type MIC strain, yet epithelial cell detachment from the basement membrane and shedding of epithelial cells in the lumen of β1-deficient glands Fig. . This phAlthough these results provide compelling evidence for the requirement of β1 integrin in the initiation phase of mammary tumourigenesis, whether β1 integrin is required for maintenance of the transformed state was unclear. To address this issue, we employed an Adenoviral Cre vector system to ablate β1 integrin expression in vitro. Using several primary cancer cell lines derived from MMTV-PyV mT tumours bearing either wild type (wt/wt) or homozygous conditional (fl/fl) alleles for β1 integrin, we achieved efficient β1 integrin ablation in the latter Fig. . We subsNext, we assessed the importance of β1 integrin during the pathological transition from non-invasive ductal hyperplasia (MIN) to invasive carcinoma in later stages of the disease Fig. . OverallE2f7, Rtel1, Myc, Ybx3 and Id3) being downregulated in β1 integrin-deficient tumours, consistent with slow tumour growth phenotype . Of particular note, β1 integrin-deficient tumours exhibited a dramatic increase in stromal content that correlated with massive deposition of collagen at later stages of tumour evolution Fig.  and epitCol, Mmp, LoxL, Cxcl12)  on pooled samples derived from early invasive carcinoma from β1 integrin-proficient  and 2609 genes per cell) and β1 integrin-deficient dormant lesions  Fig. . Uniform2) Figs.  and S7. 2) Figs. , corrobo2) Figs. .To further confirm our bioinformatic observations, IHC staining of smooth muscle actin (α-sma) on early invasive β1-deficient lesions showed a substantial increase in CAF infiltration Fig. , agreeinIt is well established that integrin crosstalk with growth factor receptors plays an important role in transducing environmental cues and in influencing numerous aspects of malignant progression , 22. We Breast cancer progression is a multistep process involving distinct pathological stages and adaptation to support tumour growth and metastatic dissemination. Although acquisition of genetic alterations underlies initiating events and loss of cellular homoeostasis, cells with perturbed homoeostasis due to such oncogenic events are often kept in check by growth arrest, cell death and host immunity. Overcoming these tumour suppressive barriers is a crucial step in permitting transition from benign lesions to malignant carcinomas. Here, we show that β1 integrin receptor signalling plays a pivotal role in restraining these initial barriers to enable tumour growth. Epithelial-specific deletion of β1 integrin results in detrimental defects in serval cellular processes including loss of cell adhesion Fig.  and cellTrp53 gene functions  alleles were obtained from Dr. Mueller and described elsewhere [Tp53 alleles were obtained from the National Cancer Institute mouse depository and described elsewhere [Generation of MIC and MMTV-PyV mT transgenic mice was described previously lsewhere . Mice wilsewhere . MMTV-relsewhere . All micSupplementary informationSupplementary table 1


The following error appeared in Section 3.5 in Ref. . InsteadWe apologize for this error.


Stenotrophomonas maltophilia causes lethal pneumonia, bacteremia, and sepsis in immunocompromised patients. As a standard of care, trimethoprim-sulfamethoxazole (T/S) is considered to be the first-line therapy for Stenotrophomonas pneumonia. Cefiderocol (CFDC) is a new parenteral siderophore cephalosporin that is transported through the outer cell membrane as a siderophore mimic that then inhibits Gram-negative cell wall biosynthesis. CFDC has potent activity in vitro against S. maltophilia; however, little is known about its in vivo activity against Stenotrophomonas pneumonia in immunocompromised hosts. We therefore studied CFDC in comparison to TS in the persistently neutropenic rabbit model of Stenotrophomonas pneumonia. This rabbit model, in contrast to conventional murine models, reflects the human pattern of infection more accurately over time.Stenotrophomonas pneumonia was established by direct endotracheal inoculation of S. maltophilia 1×1010 CFUs for tracheobronchial colonization that evolved into bronchopneumonia. Experimental groups consisted of CFDC, T/S, and untreated controls (UC). Rabbits received CFDC at 120 mg/kg IV Q8h and T/S at 5 mg/kg IV Q12h. Profound persistent neutropenia was maintained with cytosine arabinoside. Treatment was continued for 10 days.We initially studied the plasma pharmacokinetics of CFDC in non-infected and infected animals. S. maltophilia from lung tissue and BAL. This antibacterial activity coincided with significant reduction of lung weights (marker of organism-mediated pulmonary injury) in the CFDC group vs T/S and UC (p< 0.01). Survival was prolonged in the CFDC treatment group with 87% survival in comparison to that of T/S (25%) and UC (0%) (p< 0.01). There were no significant differences between non-infected and infected rabbits in CFDC pharmacokinetics. Rabbits treated with CFDC and T/S demonstrated significant decreases of residual pulmonary and BAL bacterial burden vs UC (p≤0.001). CFDC achieved full clearance of Stenotrophomonas maltophilia Pneumonia in Persistency Neutropenic RabbitsTable 1. Efficacy of Cefiderocol in Experimental S. maltophilia pneumonia in persistently neutropenic rabbits, thus laying the foundation for future clinical investigations against this lethal infection. Cefiderocol is highly active in treatment of experimental Naoki Ishibashi, MD, Shionogi, Inc. (Employee) Benjamin Georgiades, n/a, Shionogi, Inc. (Consultant) Roger Echols, MD, Shionogi (Consultant) Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support) Yoshinori Yamano, PhD, Shionogi (Employee) Thomas J. Walsh, MD, PhD (hon), Scynexis Shionogi


Varicella-zoster virus (VZV) infection may induce central nervous system complications in HIV/AIDS patients. However, it is rare to have paraplegia caused by VZV infection but no herpes zoster clinically. Asymptomatic VZV infection in HIV/AIDS patient increased the difficulty of diagnosis.We reported a 41-year-old male AIDS patient with rare asymptomatic VZV infection-induced paraplegia after his anti-retroviral therapy initiation. MRI of the spinal cord showed the morphology of the thoracic spinal cord was irregular and locally inflated. The patient was confirmed as VZV induced thoracic myelomyelitis by using the cerebrospinal fluid for metagenomic next-generation sequencing (mNGS).mNGS may contribute to disease diagnosis for asymptomatic VZV infection-induced myelitis.The online version contains supplementary material available at 10.1186/s12879-021-06611-9. Varicella-zoster virus (VZV) is also known as Human_alphaherpesvirus_3. VZV infection can cause primary varicella infection (chickenpox) in children, meanwhile a few cases will develop to herpes zoster in adults after reactivation . The inc3 copies /ml of CMV were detected in sputum on January 20th. After antimicrobial and anti-CMV treatment, the patient’s condition improved. The combined ART regimen of Nevirapine, Lamivudine, and Tenofovir was taken on January 27th, 2020. In the following days after cART initiation, he experienced weakness of lower limbs and developed paralysis of both legs and incontinence in a short time. He reported no skin lesions, fear of cold, fever, dizziness, headache, vomiting, limb convulsions, abdominal pain, diarrhea, or any other discomfort. He also reported no special medical history or trauma history. We gave him a physical examination when admission: his lower limb muscle tension was low, and the muscle strength was level 0. But the passive range of motion of all joints of the body was normal. His sensation disappeared in the sacrococcyx, buttocks, front and lateral thighs, back and medial sides, calves, and feet. However, pathological emission did not elicit.A 41-year-old male was presented in our hospital on February 15th, 2020, for bilateral lower limb paralysis for more than 10 days. In the middle of January 2020, he was admitted in the local hospital due to he had fever for more than one month. He was diagnosed with AIDS, bacterial pneumonia, and cytomegalovirus (CMV) infection in the local hospital. Seven hundreds copies /ml of CMV were detected in urine On January 16th, and 5.77 × 10+ T cell count was 35 cells/µL, white blood cells (WBC) count was 4.8 × 109/L, red blood cell (RBC) count was 3.24 × 1012/L, hemoglobin (Hb) was 90 g/L, Platelets (PLT) was 259 × 109/L, and the percentage of neutrophile granulocyte was 76.4 %. No abnormalities were found in liver and renal function tests. Screening for hepatitis B virus infection, hepatitis C virus infection, and syphilis were negative. Neither was the Mycobacterium tuberculosis (Mtb) infecting T cells test. DNA tests for CMV and Mtb in blood and CSF were negative, too. CSF examination showed the opening pressure was 60 mm H2O by lumbar puncture, the Pandy test was weakly positive, 16.00 × 106 WBC/L, and a normal level of adenosine deaminase (2.01 U/L). The biochemical indicators of CSF revealed protein concentration was 960.60 mg/L , normal level of glucose (3.59 mmol/L), and chloride concentration was 111.30 mmol/L . No bacteria or fungi was found in CSF smear and culture. MRI of the brain and electrocardiography were normal. However, MRI of the spinal cord showed the morphology of the thoracic spinal cord was irregular and locally inflated, and the signals of T1 and T2 were slightly longer . Screening results for bacteria, fungi, and parasites were negative. The results indicated the pathogen was VZV . The mNGS was done on BioelectronSeq4000 platform . After quality filtering, eliminating replicate reads, removing reads matching human genome sequence, reads were classified against the NCBI reference/representative bacterial, archaeal, and fungal genomes, as well as genomes of DNA viruses infecting humans and DNA bacteriophages. Of 25,337,336 quality filtered sequencing reads, 23,485,313 were human, 53,502 were assigned to microbe, and 3935 were mapped on Human_alphaherpesvirus_3 (VZV). The VZV genome coverage was 95.55 %, and the estimated VZV DNA concentration was 2.6 × 10After admission, he was treated with sodium phosphonate 3.0 g/3 times a day for 50 days, adjuvanted with traditional Chinese acupuncture for more than a month to control the symptom. Acyclovir 0.5 g/3 times a day was used for 22 days after he was diagnosis as VZV infection by mNGS. The MRI reexamination of the spinal on April 3rd showed that the edema of the lesion was reduce Fig. d, e. HowVZV is an enveloped DNA virus, which can invade tissues of ectodermal origin, including skin, mucous membranes, and nerve tissue. Typical VZV-related manifestations include rash and pain . VZV infThe risk of VZV infection in HIV/AIDS patients was 2.5-fold higher compared with the general population . It was 3copies/mL, and no other viruses, bacteria, fungi, or other pathogens were found. The sequencing result confirmed the diagnosis of the patient, backing by his symptoms and clinical tests. We suggest that a routine test for VZV DNA in CSF should be established for the HIV-infected patients with neurological diseases, such as PCR for VZV DNA.The clinical manifestations of the reported case didn’t include chills and fever, dizziness, headache, abdominal pain, diarrhea, or herpes zoster. Paraplegia is rarely the only manifestation of VZV infection. Therefore, the diagnosis cannot be made based on the manifestations. Here, we have made a pioneering attempt to isolate the pathogen DNA in CSF for mNGS. mNGS a high-throughput sequencing technology, has been applicated in clinical diagnosis for some unclear pathogens. With the application of mNGS, we found the sequences of VZV with an estimated viral load of 2.6 × 10The patient was treated with sodium phosphonate 3.0 g/3 times a day and acyclovir 0.5 g/3 times a day during hospitalization. Acyclovir intravenous treatment is a recommended regimen for VZV infection . Early aHere, we reported a rare case of myelitis paraplegia caused by VZV without herpes-related symptoms, and share examples of metagenomic sequencing used in clinical diagnosis of difficult diseases, to provide a reference for other similar diseases.Additional file 1. Three representative DNA sequences got from CSF by mNGS.Additional file 2. The reference sequence for sequencing analysis.


Most malignant tumours are highly heterogeneous at molecular and phenotypic levels. Tumour variability poses challenges for the management of patients, as it arises between patients and even evolves in space and time within a single patient. Currently, treatment-decision making usually relies on the molecular characteristics of a limited tumour tissue sample at the time of diagnosis or disease progression but does not take into account the complexity of the bulk tumours and their constant evolution over time. In this review, we explore the extent of tumour heterogeneity and report the mechanisms that promote and sustain this diversity in cancers. We summarise the clinical strikes of tumour diversity in the management of patients with cancer. Finally, we discuss the current material and technological approaches that are relevant to adequately appreciate tumour heterogeneity.Human solid malignancies harbour a heterogeneous set of cells with distinct genotypes and phenotypes. This heterogeneity is installed at multiple levels. A biological diversity is commonly observed between tumours from different patients (inter-tumour heterogeneity) and cannot be fully captured by the current consensus molecular classifications for specific cancers. To extend the complexity in cancer, there are substantial differences from cell to cell within an individual tumour  and the features of cancer cells evolve in space and time. Currently, treatment-decision making usually relies on the molecular characteristics of a limited tumour tissue sample at the time of diagnosis or disease progression but does not take into account the complexity of the bulk tumours and their constant evolution over time. In this review, we explore the extent of tumour heterogeneity with an emphasis on ITH and report the mechanisms that promote and sustain this diversity in cancers. We summarise the clinical strikes of ITH in the management of patients with cancer. Finally, we discuss the current material and technological approaches that are relevant to adequately appreciate ITH. Tumour heterogeneity harbours multiple layers of complexity in human malignancies. It has long been known that tumours of the same histopathological subtype commonly differ from one patient to another (inter-tumour heterogeneity) . ExacerbThe first demonstration of tumour heterogeneity has been made by histopathologists who are familiar with morphological divergence  across the tumours or between the different areas of the tumour  1,4,5,6,65,6,7,8Advances in next-generation sequencing (NGS) revealed the extent and prevalence of molecular diversity in tumours ,15. The However, the genetic perspective is insufficient to fully explain the range of phenotypic diversity in solid malignancies, given the fact that cell populations with identical genetic background can lead to distinct morphological patterns and differential responses to treatment or environmental stimuli ,21. EmerDNMT3A, IDH1, H3F3A) and inversely the identification of DNA hypomethylation or epigenetic silencing of DNA repair genes (such as MLH1 or BRCA1) that can cause genomic instability in cancer cells [The epigenome is defined as a connection between the genome and the environment. Alterations of the epigenetic machinery has been recognised as a hallmark of cancer  and may er cells . Studieser cells ,26,27. Cer cells .Transcriptome refers to all RNA species that can be found in cells; however, mRNAs are frequently the most studied. Their composition varies between cell types and tumour types and continuously evolves depending on the local conditions that are applied to cells over time. They can be explored through targeted (RT-PCR) or high throughput approaches ). A plethora of gene expression signatures have been developed in oncology for tumour classification , prognosBecause proteins directly reveal the functional mechanisms that occur in cancers and account for most of the therapeutic targets, it appears important to assess tumour heterogeneity at the protein level, which has shown growing interest. Proteomic approaches have long lagged behind those for transcriptome and genome due to technical limitations, high amounts of proteins generated from a single gene (with different isoforms and modification states) and a complex regulation of protein expression at both translational and post-translational levels . ImmunohRecognition of all these cancer-specific molecular processes as major elements in the evolution of cancers and their considerable diversity has led to the launch of cancer genomics programs by international consortia, such as The Cancer Genome Atlas consortium (TCGA) and the International Cancer Genome Consortium (ICGC). All aim to integrate the analysis of multi-omics datasets  to provide a comprehensive overview of the tumour landscape ,43,44,45The influence of the complex ecosystem in which cancer cells evolve has long been overlooked. In the last decade, cancer biology progressively shifted from a cancer cell-centric model to a more ample view, where cancer cells and their near environment are highly interrelated. The tumour microenvironment (TME) is made up of non-transformed cells  and non-cellular constituents (such as the extracellular matrix) which are shaped by cancer cells through the modification of local environmental conditions and the secretion of oncogenic signals ,47. As aTwo major and paradoxical theories have been developed to explain the installation of high degree of diversity in tumours. In 1976, Peter Nowell first described the cancer development as a continuous evolutionary process originating from a single renagade mutant cell and driven by the accumulation of stepwise somatic mutations during proliferation processes that give rise to various clones and subclones . The devIn contrast to the Darwinian clonal evolution theory, where all subclones possess tumorigenic potential, a second model proposed that only a small subgroup of cancer cells (named cancer stem cells (CSC) or tumour-initiating cells) has the capacity to generate new tumours ,69. In tIn these first models of tumour evolution, ITH was thought to gain gradually over time as the tumour grew . AlthougBranched evolutionary trajectories have been extensively described in a wide range of tumour types, such as childhood acute lymphoblastic leukaemia, clear cell renal carcinomas, pancreatic, colorectal, breast and prostate cancers . EvolutiITH has also been described in cancer cases with linear evolutionary trajectories whereby a predominant subclone outgrows at the expense of its predecessor(s) followed by incomplete selective sweeps ,77. AlthIn the last decades, the management of patients with cancer has been revolutionised by better knowledge on the molecular background in cancer development. The understanding of molecular inter-tumour heterogeneity has formed the basis of personalised medicine in diagnosis, prognosis and treatment of cancers. Notably, it has set the limits of using universal anticancer drugs and has been a major driver for the emergence of novel therapies targeting specific molecular characteristics . For mosMost of the histopathological and molecular features are not expressed homogeneously in tumour subpopulations, highlighting the fact that the analysis of a single sample may lead to diagnostic and prognostic errors and provide an incomplete view of potential vulnerabilities to treatment . The ideArchival tissue specimens commonly serve as starting material for testing if any recent sample is available. These samples can be collected many months or years previously, at the time of diagnosis or when a new lesion appears at distance. However, they cannot reliably reflect the tumour landscape over the time, considering that the tumour constantly evolves under specific microenvironmental conditions   or exposAnalysing the characteristics of a tumour or of different tumour regions allows us to define the tissue of origin but also provide insights into the molecular events that occurred sequentially or in parallel throughout the development of the tumour. In this context, evaluating ITH gives a remarkable view of the whole history of the tissue and could help to better understand cancerogenesis and develop new therapeutic strategies.The analysis of data collected by the TCGA from more than 3300 tumours across nine tumour types revealed that ITH has prognostic utility . High deEmerging omics technologies have shown their interest in recent studies to analyse all types of ITH. However, multi-omics analyses are still far from standard-of-care, considering their cost, their low spread in clinical labs and the need for powerful data storage options for these big data. The analysis of only few histomolecular biomarkers in tumours is still the reference and may lead to misinterpretation.Considering the huge amounts of data generated from multi-omics approaches and their high complexity, there is a considerable need to develop automation tools able to provide an integrative analysis of the multiple layers of heterogeneity without any expert intervention. Last advances in artificial intelligence and machine learning models allowed us to better predict cell subtypes and infer their proportions in tumours and TME based on their inherent multi-omics characteristics. These approaches have the potential to integrate both molecular and histopathological imaging data to refine tumour heterogeneity in the spatial context and go beyond what can be distinguished by routine microscopy observations ,91. HoweAlthough the notion of ITH and its impact on therapeutic response is now well documented in research studies, ITH determination is rarely taken into account in current clinical decision making that mostly relies on short-term treatment efficacy and the detection of resistance mechanisms to adapt the treatment and forestall disease relapse. However, capturing ITH could aid in developing novel strategies to provide long-term drug response and minimise the emergence of resistance mechanisms . For exaDecoding tumour heterogeneity is a major clinical challenge, considering that it immensely contributes to cancer progression, treatment failure and emergence of drug resistance. Emerging technical and sampling strategies have been developed in order to deeply characterise tumour complexity and clonal architecture, including single-cell profiling, multi-region sampling, representative sampling and longitudinal analysis of liquid biopsy samples .Most studies that aim at profiling ITH and clonal evolution were based upon standard bulk-cell methods , that reSingle-cell approaches (SCA), based on the analysis of a high number of cells individually, gained popularity in the last few years, as they can unambiguously reconstruct tumor subclonality. SCA provided a valuable opportunity to detect slight cell-to-cell variations and perform cell-type clustering, determine ITH and TME composition at high resolution level, and evaluate the cellular evolutionary relationships ,100,101.For decades, the tissue biopsy sample remains the reference practice for tumour testing. However, tissue biopsy sampling implies an invasive and putatively morbid procedure. Further, it may be unfeasible in certain cases, as the tumour site is not accessible. Moreover, the analysis of a unique limited tumour tissue sample provides by nature only a spatiotemporal snapshot of the tumour. The input material for routine molecular characterisation generally represents as little as 0.0005% of the tumour bulk, making it inappropriate to fully capture the complexity of the whole disease .BAP1 mutation or VHL wild-type status, lower ITH and high genomic instability. Contrariwise, attenuated progressive diseases  preferentially harboured “PBRM1 → SETD2” or “PBRM1 → PI3K” evolutionary trajectories, high ITH and low genomic instability in primary tumours. This example illustrates well the interest of multi-region sampling to better decipher the impact of ITH and genetic evolution on cancer progression. However, such strategy seems impractical in routine care given the invasive nature of the process and the marginality of tissue rebiopsy during the disease. Moreover, the analysis of multiple samples in parallel implies tedious and costly procedures that cannot be easily performed on standard tumour biology platforms.Procuring multiple samples of the tumour for molecular analysis represents one way to circumvent this limitation ,113,114.Pooling DNA/RNA extracts from multiple anatomically distinct regions of the tumour before sequencing could represent an option to reduce the cost, while optimising the variant detection rates as well as the determination of mutation prevalence and clonal clustering in the tumour mass compared to single-biopsy sampling . NeverthRepresentative sampling has been proposed by Litchfield et al. as a more realistic approach for clinical routine . RepreseEmerging studies resorted to the analysis of tumour samples collected from patients during autopsy to tackle the encountered limitations of sampling in living patients ,123. SucLiquid biopsy (LB), based on the analysis of tumour markers circulating in body fluids (including circulating tumour cells (CTCs), circulating tumour nucleic acids , circulating nucleosomes and tumour-derived extracellular vesicles), is emerging as a promising alternative to tumour tissue biopsy. Numerous studies already demonstrated its interest as a minimally invasive and easily repeatable tool for cancer screening, diagnosis, prognosis, treatment stratification, minimal residual disease (MRD) evaluation, disease monitoring and early detection of resistance mechanisms ,132,133.CTCs have been less extensively studied than ctDNA but research studies on CTCs have been on the rise for a few years. CTCs were found to be heterogeneous at genetic and phenotypic levels and evolve over the time as the tumour progresses . As CTCs6–1:108 of all blood cells) [CTC-based approaches imply a critical step of CTC isolation from blood samples prior to single- or multi-omics analysis . CTC analysis based on single-cell approaches has the advantage of providing representative information on ITH at higher resolution level . CTC isod cells) ,136,146.d cells) . The Celd cells) . Howeverd cells) .Cell-free DNA (cfDNA) is shed in body fluids by malignant and non-malignant cells through different biological processes, including apoptosis, necrosis and spontaneous active release (incorporated or not in extracellular vesicles or nucleoproteic complexes) . CtDNA rIn the Rep-Seq study, the clinical interest of plasma samples drawn at different time points was evaluated to delineate ITH in ccRCC compared to Rep-Seq and multi-region sampling . AlthougIn the same way, the NSCLC TRACERx study aimed at assessing the clinical utility of ctDNA to track cancer evolution in 100 patients with resectable Non-Small Cell Lung Cancer (NSCLC) . Multi-rDNMT3A, TET2, ASXL1), spliceosome proteins , DNA damage response regulators  or cell signalling pathway actors  [One challenge of using plasma samples in cancer care is the fragmentation of ctDNA molecules and their high dilution in cfDNA shed by non-malignant cells . Recent2, GNAS) . In such2, GNAS) ,161.Tumour heterogeneity appears multifaceted and pervasive across cancer types and limits the diagnosis, prognosis and predictive value of tumour biomarkers. Although all levels of tumour variability lead to challenges in oncology, ITH is of particular interest considering its major consequences on cancer progression, metastatic dissemination, targeted therapy failure, drug resistance and recurrences. The clinical evaluation of tumour diversity should be a prerequisite for treatment-decision making; however, the information obtained routinely from a single-site tumour tissue biopsy is not sufficient to fully reflect the bulk tumour and its heterogeneity. Novel approaches are emerging to better capture ITH. They present differential advantages and limitations that have to be considered in order to select the most appropriate strategy depending on the clinical and biological context. Single-cell approaches can reliably define extensive tumour taxonomy but suffer from their cost and technical challenges. Multi-region sequencing represent appealing approaches to decipher the clonal architecture of the tumours and understand the biology of metastasis; however, Rep-Seq appears as a much more practical alternative for clinical routine. Liquid biopsy offers the advantages to more easily reveal spatial but also temporal ITH. The development of patient-specific cfDNA-based targeted assays notably holds great promise for the longitudinal tracking of genomic evolution during the course of the disease at more moderate cost.


Glioblastoma (GBM) is the most common primary adult brain tumour with a median overall survival (OS) of 12–15 months. Molecular characterization of multiple immunooncology targets in GBM may help target novel immunotherapeutic strategies. We used NanoString GeoMx® Digital Spatial Profiling (DSP) to assess multiple immunooncology protein targets in methylated versus unmethylated IDH-wild-type glioblastoma.  NanoString GeoMx® DSP technology uses multiple primary antibodies conjugated to indexing DNA oligos with a UV photocleavable linker. Tissue regions of interest (ROIs) are selected with bound fluorescent antibodies; oligos are released via a UV-mediated linker and quantitated. We used DSP multiplex analysis of 31 immunooncology proteins and controls , CD45, Pan Cytokeratin, MS4A1/CD20, CD45RO, PD1, CD3, beta-2 microglobulin, VISTA, Bcl2, GZMB, PTEN, beta-catenin, CD56, Ki-67, STAT3, AKT, p-Akt, S6, Histone H3, IgG Rabbit control, and Mouse IgG control) from ROIs in a cohort of 10 IDH-wild-type glioblastomas (5 methylated and 5 unmethylated). An nCounter platform allowed quantitative comparisons of antibodies between ROIs in MGMT methylated and unmethylated tumours. Mean protein expression counts between methylated and unmethylated GBM were compared using technical and biological replicates.  The analysis showed 10/27 immunooncology target proteins were significantly increased in methylated versus unmethylated IDH-wild-type glioblastoma tumour core  <0.1 by Benjamini–Hochberg procedure).  NanoString GeoMx® DSP was used to analyse multiple immunooncology protein target expression in methylated versus unmethylated IDH-wild-type glioblastoma. In this small study, there was a statistical increase in CD4, CD14, CD68, CD8A, B7-H3, PDL-1, CD19, FOXP3, CD44, and STAT3 protein expression in methylated versus unmethylated GBM tumour core; however, this requires larger cohort validation. Advanced multiplex immunooncological biomarker analysis may be useful in identifying biomarkers for novel immunotherapeutic agents in GBMs. Opdivo in patients with first recurrence of glioblastoma multiforme (GBM), did not meet its primary endpoint of improved overall survival over bevacizumab monotherapy highlighting the need for more detailed analysis of immunooncology biomarkers within GBM in order to better target novel immune therapeutics , MGMT methylated tumour core mean normalised value (MGMTmc) = 9.19 [1.00]), and CD45RO isoform (MGMTuc = 7.48 [0.50] and MGMTmc = 8.29 [0.81]) which is in keeping with low immune infiltration irrespective of MGMT methylation status within IDH-wild-type GBM. The mean normalised count values of T lymphocyte marker CD3 are low (MGMTuc = 8.04 [1.02] and MGMTmc 8.90 [0.61]) and T-helper cell marker CD4 (MGMTuc = 9.58 [1.01] and MGMTmc = 11.22 [0.65]), which may indicate very low levels of T-cells and T helper cells, respectively. Both B-cell markers CD20 (MGMTuc = 6.47 [0.53] and MGMTmc = 7.26 [0.63]) and CD19 (MGMTuc = 6.66 [0.63] and MGMTmc = 7.77 [0.74]) are reduced which also may indicate a very low number of B-lymphocytes within cases. Mean normalised values for cytotoxic T-cell marker CD8A (MGMTuc = 6.90 [0.66] and MGMTmc = 8.30 [0.78]) may indicate low levels of cytotoxic T cells. By comparison, GZMB  shows elevated values. GZMB is a serine protease commonly found in the granules of cytotoxic T-cells and NK cells, and although difficult to be certain, the mild increased values may represent a difference in NK cells. We observed comparatively low normalised mean values of PD-1 (MGMTuc = 7.43 [0.44] and MGMTmc = 8.08 [0.73]) in keeping with overall comparatively low T-cell-associated proteins in our cohort.p-values for MGMT comparison in tumour core (column 2) and margin (column 3) and for comparing core against margin in methylated (column 4) and in unmethylated (column 5). Overall there was a statistically significant difference between methylated and unmethylated tumour cores, but not tumour margins. GeoMx DSP shows 10/27 immunooncology target proteins were significantly increased in methylated versus unmethylated IDH-wild-type glioblastoma tumour core but not margin  <0.1, by Benjamini–Hochberg procedure). In particular, there was a statistically significant increase in CD4, CD14, CD68, CD8A, B7-H3, PDL-1, CD19, FOXP3, CD44, and STAT3 protein expression in methylated versus unmethylated GBM tumour core (see core see . There wp = 0.025) (see p = 0.023); MGMT methylated tumour core counts: 14.07 [1.02] compared to MGMT unmethylated GBM (tumour core: 12.35 [0.87]). V-domain immunoglobulin suppressor of T-cell activation VISTA is homologous to PD-L1 and a suppressor of T-cell activation, synergising with PD-1. By contrast, VISTA is not statistically increased in MGMT methylated tumour cores compared with unmethylated tumour cores of GBMs (p = 0.153); MGMT methylated tumour core (11.94 [0.70]) and MGMT unmethylated tumour core (11.42 [0.77]) although mean normalised counts are increased.In particular, in our cohort, checkpoint biomarker PD-L1 is statistically increased in MGMT methylated tumour cores compared with unmethylated tumour cores of GBMs (025) see . The cheIn this paper, we used NanoString GeoMx® Digital Spatial Profiling (DSP) technology to analyse multiple immunooncology targets in methylated against unmethylated IDH-wild-type GBM tumours. NanoString GeoMx® Digital Spatial Profiling (DSP) technology has the theoretical ability to analyse a potential of 800 protein targets, therefore allowing in-depth analysis of panels of tissue-based prognostic and predictive biomarkers, in-depth monitoring of response to therapies, and more rapid identification of targets of disease. The small sample of this technology feasibility study requires further validation of results in larger cohorts.The mean log 2 housekeeping normalised values by location and MGMT status for all 27 immune protein targets ranged from 6.47 to 19.26 in our study . NanoStrp = 0.03) between MGMT methylated and unmethylated tumour core, although overall counts are low (MGMTuc = 7.02 [0.51] and MGMTmc = 7.94 [0.73]). In a recent study of 186 IDH-wild-type glioblastoma patients, consecutively treated with radiochemotherapy, the presence of FoxP3 + cells was associated with a better overall survival  ) , althoug4–0.98]) . Additio4–0.98]) . In anotduration . The degp = 0.025). CheckMate 143 also showed evidence of increased PD-L1 expression in a cohort 27/40 (68%) glioblastoma raising the possibility of PD-L1 inhibitor (nivolumab) use in clinical practice [PD-L1 on glioblastoma cells interacts with PD-1 normally expressed on the surface of T-cells and this interaction may suppress T-cell activation. In our cohort, PD-L1 expression is statistically increased in MGMT methylated tumour cores compared with unmethylated tumour cores of GBMs (practice . Howeverpractice . One expp = 0.023). A similar expression pattern has recently been reported by a study (n = 994) by Wang et al. who demonstrated B7-H3 upregulation in high-grade glioma and hence lower overall survival [In addition, we observed higher overall mean normalised values of the checkpoint molecule B7-H3 (CD276) irrespective of methylation status; MGMT methylated  compared to MGMT unmethylated GBM . We also noted a statistical increase of B7-H3 in MGMT methylated tumour core B7-H3 (CD276) against MGMT unmethylated tumour core , which is homologous to PD-L1, is a suppressor of T-cell activation, synergising with PD-1 , 33. In Although in our small study there was a statistically significant increase in CD4, CD14, CD68, CD8A, B7-H3, PDL-1, CD19, FOXP3, CD44, and STAT3 protein expression in methylated versus unmethylated GBM tumour core see , we needThe small size of our cohort limits interpretation. In this study, the four main sources of variation in using DSP are nCounter quantification, ROI background, ROI size, and ROI cellularity . SectionAnother disadvantage of DSP technology is that it only permits targeted discovery, and a selection of targets is required to be included in the cocktail of antibodies, rather than “pure discovery” . Other tIn our experience, careful consideration of the experimental design and normalization process, with simultaneous in-depth understanding of DSP normalization may optimise results. In this small study, there was a statistical increase in CD4, CD14, CD68, CD8A, B7-H3, PDL-1, CD19, FOXP3, CD44, and STAT3 protein expression in methylated versus unmethylated GBM tumour core; however, this requires larger cohort validation. Advanced multiplex immunooncological predictive biomarker analysis may underpin a future personalised medicine approach in the use of targeted immunotherapeutic agents in glioblastoma.