Soft micro‐ and nanostructures have been extensively developed for biomedical applications. The main focus has been on multifunctional composite materials that combine the advantages of hydrogels and colloidal particles. Magnetic microgels and nanogels can be realized by hybridizing stimuli‐sensitive gels and magnetic nanoparticles. They are of particular interest since they can be controlled in a wide range of biological environments by using magnetic fields. In this review, we elucidate physical principles underlying the design of magnetic microgels and nanogels for biomedical applications. Particularly, this article provides a comprehensive and conceptual overview on the correlative structural design and physical functionality of the magnetic gel systems under the concept of colloidal biodevices. To this end, we begin with an overview of physicochemical mechanisms related to stimuli‐responsive hydrogels and transport phenomena and summarize the magnetic properties of inorganic nanoparticles. On the basis of the engineering principles, we categorize and summarize recent advances in magnetic hybrid microgels and nanogels, with emphasis on the biomedical applications of these materials. Potential applications of these hybrid microgels and nanogels in anticancer treatment, protein therapeutics, gene therapy, bioseparation, biocatalysis, and regenerative medicine are highlighted. Finally, current challenges and future opportunities in the design of smart colloidal biodevices are discussed. BMP‐2bone morphogenetic protein 2LCSTlower critical solution temperatureMGmicrogelmiRNAmicroRNAMMGmagnetic microgelMNGmagnetic nanogelMNPmagnetic nanoparticleMPImagnetic particle imagingMRImagnetic resonance imagingNGnanogelNIPAMN‐isopropyl acrylamideNPnanoparticlePEGpoly(ethylene glycol)PNIPAMN‐isopropyl acrylamide)poly (PNIPAM) is the most studied polymer, and it shows a well‐characterized coil‐globule transition around a critical temperature.For synthetic macromolecules, poly network, the resulting material is called a hydrogel.While hydrogels are liquid‐like solutions in which water‐dissolved polymer chains tend to maintain maximum spatial distribution to minimize the mixing free energy , they are also solid‐like since the cross‐linked polymer matrix is elastic. They retain their elastic free energy since they swell, but they do not fully dissolve in aqueous environments because of the cross‐linked polymer networks and maintain their 3D structures.Generally, for a hydrogel in a swollen state, the balance between polymer–water mixing free energy and the elastic free energy of the cross‐linked polymer network determines the dimensions of the hydrogel at equilibrium. If hydrogel‐containing polymers are responsive to external stimuli, the hydrogel can exhibit a phase transition by swelling and deswelling (shrinking)Diffusion and convection are core mechanisms responsible for the use of hydrogels in biomedical applications, especially for controlled drug release.We first consider diffusion‐controlled deliveryThe second case is a swelling‐controlled release system, which refers to a dynamically swelling hydrogel matrix in which drugs are uniformly dispersed.Convective transport phenomena are strongly implicated in studies of the magnetic drug delivery in vivo. First, the body‐injected magnetic nanodrug carriers are subjected to a blood or lymph flow through the peripheral microvascultures.Second, instead of depositing drug carriers at a target site using a static magnetic force, wireless electromagnetic manipulation, and control of the motion of colloidal micropropellers by applying rotating magnetic fields can promote the transport of nanodrug carriers via augmented local fluid convection.Finally, magnetic field‐induced actuation of drug‐loaded ferrogels has been highlighted as an efficient method for on‐demand and remotely controllable drug delivery devices.Since magnetic fields permeate bulk tissues and organs, MNPs have a strong advantage over other types of inorganic biomaterials in biomedical applications.Excellent reviews of the general properties of MNPsIn a discussion of MNPs, it is instructive to treat them as a magnetic macrodipole; we ignore the detailed distribution of atomic spins in the NPs and consider an entire NP as a magnetic dipole with dipole moment m (ampere meter square), which is the sum of the projection of all atomic spins onto the mean direction. The dipole moment can be a function of the applied field B (tesla) and generally increases with the NP size. Such a macrodipole experiences a torque in a uniform field, and an additional force in a nonuniform magnetic field . Figure In most applications, it is desirable that MNPs have minimal remanence. In such a case, the MNPs do not attract each other when there is no external field, and it is convenient to use them in the form of a suspension. Since only MNPs with an easy axis show remanence, it is important to produce spherical MNPs. It is difficult to avoid a preferred direction of magnetization, which is intrinsic to atomic crystals. There are methods to suppress remanence, using superparamagnetism and magnetic domains (see Supplementary Information).When the field is swept, the atomic spins in the NP can follow either through the rotation of the entire MNP by a torque or because of the rotation of the spins in the MNPs.2mB (torque integrated over angle). In the case of pure Néel relaxation, the maximum loss is 4mBc (maximum area under the hysteresis loop). Since Néel relaxation is considerably faster than Brownian relaxation, the Néel loss is the maximum loss. Applying fields higher than the saturation field , are proportional to B2.B2f).For nonremanent MNPs, the relation between the moment and the field around Magnetic hydrogels can be formed by confining water‐based suspensions of magnetic particles to the swollen 3D network of polymers.Depending on the gel‐MNP hybridization type, MMGs and MNGs may fall into one of the following categories Figure :Hollow gel‐shells filled with magnetic fluidsMGs or NGs with an MNP‐embedded or coated outer layerMGs embedded with MNPsSingle MNPs coated with a hydrogel layerDuring the designing of these hybrids,Another key aspect in the physical design of magnetic gel hybrids is the time scale of gel swelling and deswelling in response to the applied magnetic fields. In particular, for repeated volume phase transition of MNP‐laden thermoresponsive hydrogels,In Part I, we presented the background theory for MGs, NGs, MNPs, and their hybrids. In the second part of this review, we summarize recent progress in the application of these hybrids in biomedical fields, including controlled drug release, cancer therapeutics, protein and gene delivery, and bioseparation and biocatalysis.There are various strategies to exploit the unique physical properties of MNP‐hybridized MGs/NGs in biomedical microdevices.Let us consider possible scenarios for such platforms. After injection into the body, drug‐loaded MMGs and MNGs can be guided by magnetic forces to specific locations in vivo.Drug delivery is currently the main application area for MMGs and MNGs. However, they can be applied in many other important frontier fields in precision medicine, such as cancer chemotherapy, protein and gene therapeutics, tissue engineering, and regenerative medicine, apart from bioseparation and biocatalysis. Notably, all these applications are mutually related in terms of engineering principles and practical uses. In tissue engineering, for instance, one may regenerate completely different complex tissue architectures depending on the extent of spatiotemporal control of the morphogen concentration within in vitro cellular architectures or in vivo native tissues provided by the drug delivery technique employed.There have been several physicochemical investigations on MMGs and MNGs with potential for a wide range of drug delivery and controlled release applications. These studies focused on synthesis, structure characterization, and stimuli‐controlled responsivity with or without the consideration of model drug release effectiveness.3O4) NP‐containing mesoporous silica particles coated with a gel of NIPAM and N‐hydroxymethyl acrylamide copolymer act as thermosensitive microspheres.For example, it has been reported that magnetite , and they have shown in vitro effectiveness for HUVEC and HT29 cell linesIn particular, Sunderland et al.3O4 NPs entrapped in the PNIPAM network.3O4 core coated with PEG copolymer and PNIPAM gel layers for the delivery of methotrexate,3O4 NPs with a layer of PNIPAM copolymer gel for cisplatin release,High‐frequency magnetic field‐responsive PNIPAM‐based MGs have also shown controlled release capability for anticancer curcumin through induction heating of Fe3O4 NPs with a porous carbon shell in which fluorescent carbon dots were embedded and (2) an outer PNIPAM NG layer in which drug (curcumin) molecules were loaded. This system exhibited responsivity caused by the heating of the superparamagnetic NPs by alternating magnetic fields and the heating of the carbon shell by near‐infrared light. Subsequent deswelling of the PNIPAM gel layer enabled drug release and photoluminescence tuning, leading to selective imaging and cytotoxicity of B16F10 cells in vitro. From a different perspective, another type of dual responsive MNGs was developed.g‐poly(N‐vinylcaprolactam) NG that was designed to release preloaded doxorubicin in response to high‐frequency magnetic fields. This NG platform showed synergic cytotoxicity for breast cancer cells for both hyperthermia and chemotherapy. A recent interesting study developed a neutrophil‐inspired magnetic peptide NG loaded with Fe3O4 NPs and chloroperoxidase.2O2 levels in cancer cells and that subsequently, chloroperoxidase converted H2O2 into singlet oxygen. This NG facilitated synergic anticancer therapy involving a combination of magnetic hyperthermia and enzymatic treatment.For the achievement of multifunctionality, a more complex hybrid NG platform was developed,The controlled delivery of proteins3O4 core coated with a polyacrylamide gel network in which enzymes  were encapsulated. They showed the possibility of using the complex for static‐field controlled delivery of proteins and subsequent cellular metabolic manipulation. Another promising attempt involved a physically cross‐linked polysaccharide NG, “chaperone,” that was hybridized with oleic acid‐coated Fe3O4 NPs through hydrophobic interaction.For protein delivery, Lin et al.co‐acrylamide) chains were trapped and ferrofluids were confined. Upon applying an alternating magnetic field, the hybridized gelatin matrix collapsed because of internal magnetothermal effects and the preloaded model drug  molecules were released through convective transport. On the basis of the biocompatibility and enzymatic degradability of this MG platform, potential applications in tissue engineering can be expected.Furthermore, a hybrid MG device that combines biodegradability and magnetic field‐responsivity has been developed.3O4 NPs, and the heparin component facilitated the adsorption of bone morphogenetic protein 2 (BMP‐2) with high efficiency. Guided by a static magnetic field in vitro, the biopolymeric MNGs promoted the human osteoblast MG‐63 viability by releasing the loaded cell growth factor BMP‐2. The protein release was considered to be triggered by enzymatically induced swelling and degradation. This is one more example of a biocompatible NG system that can be applied to cartilage and bone tissue regeneration.A nanoscale platform for in‐depth tissue engineering applications was developed by Fan et al.3O4 NPs that could be degraded for renal clearance. The model drug dextran was loaded in these MNGs. In cell culture experiments, the ligand‐conjugated MNGs showed significant cellular uptake by human umbilical vein endothelial cells, while macrophage uptake was low. Their possible use for the delivery of DNA and small interfering RNA (siRNA) was discussed. Alternatively, a hybrid MNG based on 2‐vynylpyridine‐co‐divinylbenzene copolymer could possibly be used for pH‐sensitive delivery of siRNA molecules.The efficiency of intracellular delivery of nucleic acids can be significantly enhanced by using magnetic force. Among several in vitro studies, the experiment of Deka et al.3O4 NP‐containing amphiphilic NG and a pheochromocytoma PC12 cell‐derived exosome loaded with mRNA and miRNA. This nanohybrid RNA vehicle could be efficiently delivered into adipose‐derived mesenchymal stem cells under a static magnetic field to induce cellular differentiation in neuron‐like cells.A recent study of Mizuta et al.Hydrogel composites have been extensively investigated for application to bioseparation2O4 NPs coated with a polyacrylamide gel layer whose surface was functionalized with amine groups. By using this platform, they covalently immobilized a digestive proteolytic enzyme (α‐chymotrypsin). This showed the potential of the platform for use in the magnetic separation of enzymes. Another type of magnetic adsorbent MG for high‐efficiency protein separation under high‐gradient static fields was developed by Turcu et al.3O4 NPs coated with a hydrogel shell of polyacrylic acid, poly‐N‐isopropopylacrylamide, and poly‐3‐acrylamidopropyl trimethylammonium chloride. The work of Ghaemy and Naseri3O4 NPs could release a nonsteroidal anti‐inflammatory agent (sodium diclofenac) in a controlled manner. These hybrid NGs showed potential for use in magnetically assisted separation of proteins such as albumin.For enzyme immobilization and magnetic separation through static fields, Hong et al.3O4/Fe2O3 NPs. Under alternating magnetic fields, this hybrid hydrogel membrane was shown to function as a switchable biomacromolecular filter. In this system, the deswelling PNIPAM NGs acted as magnetoresponsive valves for dextran separation, which was triggered by induction heating of the coembedded magnetic NPs.In a different concept, Lin et al.3O4 NPs were synthesized in situ. The gel surface was functionalized by cancer cell‐specific antibodies to capture the cells in a blood specimen and separate them under a local magnetic field. In another study, a clay MG containing iron oxide NPs and bacterial plasmids was developed for repeatable cell‐free protein synthesis and magnetic separation.Recently, MMG‐based isolation of breast cancer cells in the whole blood was demonstrated by Seyfoori et al.The integration of stimuli‐responsive MGs with a magnetic biomanipulation system is another recent advancement. A gold‐nanorod‐embedded double network of alginate and PNIPAM was used to fabricate a near‐infrared‐responsive MG, which was incorporated as a mechanically functional component in a magnetic microgripper.3O4 NP surface with oppositely charged enzymes. This MNP‐enzyme complex was subsequently coated with a cross‐linked polyacrylamide gel layer. Enzymes of various biological origins  were tested for intragel encapsulation, and a wide range of magnetic field‐assisted nanobiocatalytic applications was expected, in addition to applications to protein delivery and cellular metabolic manipulation.For biocatalysis, Lin et al.3O4 NPs and dual enzymes  were incorporated in a glycol chitosan gel matrix. This NG acted as a dual‐modality probe for (1) MRI by means of the superparamagnetic NPs and for (2) ultrasound imaging owing to O2 bubble generation by an enzymatic reaction with reactive oxygen species. These features facilitated the biocatalytic sensing of pathological microenvironments in vivo. Along the same lines, Wu et al.3O4 NPs were coated through free‐radical polymerization triggered by the cascade reaction of glucose oxidase and horseradish peroxidase. The encapsulated bienzyme components were covalently immobilized in the gel network, minimizing the diffusive dissipation of intermediate hydrogen peroxide between both the enzymes, which maximized the reaction efficiency. This system was designed to be suitable for colorimetric glucose sensing.Wang et al.In addition to the above studies, there have been other investigations on these topics, and their results may have the potential for use for health and hygiene purposes.Tissue regeneration is one of the most promising frontiers of MMG research. Over the past two decades, a variety of functional MGs and NGs have been developed for use in regenerative medicine.2O3‐NP‐entrapped MGs in self‐assembled structures of cell‐encapsulated MGs. A cell‐tracking study reported an alternative strategy for tissue regeneration involving mesenchymal‐stem‐cell‐internalizable MMGs.In addition to field‐guided artificial tissue construction, self‐assembly of mesenchymal‐stem‐cell‐laden alginate MGs has been achieved through surface modification with binding pair molecules, such as biotin and streptavidin.In the first part of this paper, we described the physical engineering principles that are essential for the high‐precision design of MMGs and MNGs, ranging from stimuli‐responsive behavior of hydrogel network, diffusion, and transport phenomena, to colloidal nanomagnetism. In the second part, we related those principles to the biomedical functionality of MMG/MNG systems, summarizing recent progress on these systems and their biomedical and clinically relevant applications. New promising platforms have been reported, and they facilitate on‐demand drug release and show remote controllability under static and alternating magnetic fields. Moreover, extensive research on smart gel synthesis and organic/inorganic functionalization techniques has accelerated the advent of new‐generation hydrogel materials.Despite the rapid growth of the field, the range of applications does not meet the demands of clinical practices, indicating that the current status of MMG/MNG development is not mature. In order to overcome this barrier and facilitate clinical translation, more predictable and effective systems should be developed based on the precisely tunable properties of nanobio interfaces in vivo. By harnessing more elaborate biofunctionality, including biodegradability and cellular interactions, we could facilitate the introduction of MMGs/MNGs into the clinic. In addition, potential toxic effects of magnetic nanomaterials should be identified to facilitate measures for enhancing their safety. The ultimate objective should be the development of in vivo self‐regulating nanobio‐gel systems that can report local physiological information (via fluorescence or magnetic resonance) and can be magnetically controlled by field manipulators outside the body or 3D complex tissue cultures.The authors declare no financial or commercial conflict of interest.https://publons.com/publon/10.1002/btm2.10190.The peer review history for this article is available at Appendix S1: Supporting InformationClick here for additional data file.


Active surveillance has been proposed for patients with oesophageal cancer in whom there is a complete clinical response after neoadjuvant chemoradiotherapy (nCRT). However, endoscopic biopsies have limited negative predictive value in detecting residual disease. This study determined the location of residual tumour following surgery to improve surveillance and endoscopic strategies.The present study was based on patients who participated in the prospective preSANO trial with adenocarcinoma or squamous cell carcinoma of the oesophagus or oesophagogastric junction treated in four Dutch hospitals between 2013 and 2016. Resection specimens and endoscopic biopsies taken during clinical response evaluations after nCRT were reviewed by two expert gastrointestinal pathologists. The exact location of residual disease in the oesophageal wall was determined in resection specimens. Endoscopic biopsies were assessed for the presence of structures representing the submucosal layer of the oesophageal wall.In total, 119 eligible patients underwent clinical response evaluations after nCRT followed by standard surgery. Residual tumour was present in endoscopic biopsies from 70 patients, confirmed on histological analysis of the resected organ. Residual tumour was present in the resection specimen from 27 of the other 49 patients, despite endoscopic biopsies being negative. Of these 27 patients, residual tumour was located in the mucosa in 18, and in the submucosa beneath tumour‐free mucosa in eight. One patient had tumour in muscle beneath tumour‐free mucosa and submucosa.Most residual disease after nCRT missed by endoscopic biopsies was located in the mucosa. Active surveillance could be improved by more sampling and considering submucosal biopsies. Remnant cancer missed by endoscopic biopsies was located in the mucosa in two‐thirds of patients with residual disease after neoadjuvant chemoradiotherapy (nCRT). One‐third of patients had residual disease in the submucosa underneath a tumour‐free mucosa. The yield of biopsies in active surveillance after nCRT could be improved by sampling larger areas of oesophageal mucosa and using biopsies targeting the submucosa.What lies beneath After neoadjuvant chemoradiotherapy (nCRT) for locally advanced oesophageal cancer, nearly one‐third of patients have a pathologically complete response The preSANO trialThere is a need to investigate how endoscopic surveillance and biopsy protocols can be optimized to minimize sampling errors in this patient population. The aims of this study were to assess the exact location of undetected residual disease after nCRT and to determine the depth of bite‐on‐bite biopsies.2 body surface area) for 5 weeks concurrently with 41·4 Gy radiotherapy in 23 fractions. Patients for whom surgical resection specimens were not available for review were excluded from analysis. All patients with detected residual disease from the initiating centre  were included consecutively and comprised the control group. This group was included to gain more insight in the location of residual tumours that could be detected during CREs. Patients with undetected residual disease from all centres were defined as the study group. The study protocol was approved by the medical ethics committee of Erasmus MC . All patients provided written informed consent for analysis and publication. The study was registered with the Netherlands Trial Register (NTR4834).The present study included patients who participated in the prospective preSANO trialAll patients underwent baseline clinical staging using endoscopic biopsies, endoscopic ultrasonography (EUS) with fine‐needle aspiration (FNA) of suspected relevant lymph nodes, and PET–CT. During baseline endoscopy, the distance between the incisors and upper and lower border of the primary tumour was measured. The quadrants of the oesophagus that involved tumour were specified as well. After completion of nCRT, patients underwent one or two clinical response evaluations (CREs). The first (CRE‐1) was planned 4–6 weeks after completion of nCRT, and included endoscopy with biopsies. During CREs, white‐light endoscopy was used with either regular or bite‐on‐bite biopsies using standard‐sized forceps. If no lesions were visible, at least four random biopsies were taken from the original location of the primary tumour described at baseline endoscopy. Additionally, biopsies were taken from all suspected lesions and from the borders of all ulcers. When residual vital tumour cells were detected, patients underwent PET–CT to exclude distant metastases, before oesophagectomy was performed. When no tumour cells were detected during CRE‐1, a second examination (CRE‐2) was planned 10–14 weeks after completion of nCRT. CRE‐2 consisted of PET–CT followed by endoscopic biopsies and EUS with FNA of all suspected lymph nodes. When distant metastases were detected, patients were referred for palliative care. Patients were considered to have achieved a cCR if no residual vital tumour cells were detected during CRE‐1 and CRE‐2 in endoscopic biopsies and in EUS‐guided FNA cytology. In the preSANO trial, all patients underwent standard oesophagectomy. In the present study, undetected residual disease was defined as all residual tumour with TRG 2–4  in the resection specimen that was not detected during CRE‐1 and CRE‐2.Resection specimens and endoscopic biopsies were reviewed in all patients with residual tumour that was not detected by endoscopy during CRE (study group). The exact location of the residual tumour in the resection specimen was determined and compared with that from the control group of patients who had residual tumour detected endoscopically during CRE. Review of the resection specimens and biopsies was done independently by two experienced upper gastrointestinal pathologists. All resection specimens were processed and sampled using a standard protocolFig. Fig. Tumour cells in the resection specimen were considered vital if their cytomorphological integrity was intact. A microscopically radical resection (R0) was defined by the absence of cancer cells at the proximal, distal and circumferential margin of the resection specimen. The resection specimen was scored for overall TRG using the modified Mandard scoreTo gain insight into the depth of tissue sampled by endoscopic biopsies, and the potential to detect mucosal and submucosal tumours, all endoscopic biopsies taken during CRE‐1 and CRE‐2 were reviewed for both the presence of mucosal and submucosal tissue, and the presence of vital tumour cells in the submucosal tissue if applicable. The presence of submucosal tissue was defined as described above. If only mesenchymal or ulcerative tissue was detected, the nature of the tissue present in the biopsy was defined as uncertain; otherwise, the tissue was defined as mucosal.t test or Mann–Whitney U test was used for analysis of continuous variables, and χ2 or Fisher's exact test for comparison of categorical data . P < 0·050 (2‐sided) was considered statistically significant. All statistical analyses were done using the tableone package of R version 3.5.1 .Descriptive statistics were used to describe baseline characteristics. Continuous variables are reported as median (i.q.r.). Student's Fig. Table Table Between 2013 and 2016, 207 patients underwent nCRT, of whom 119 had one or two CREs followed by standard surgical resection. Tumour cells were detected in 70 of 119 patients during CREs, including 32 patients from the Erasmus MC – University Medical Centre who served as control group. No tumour cells were detected at CREs in 49 of 119 patients, of whom 22 had a pCR in the resection specimen. Vital tumour cells were identified in the resection specimen, which had not been detected in the endoscopic biopsies, in 27 of 49 patients (study group)  Table 2. Only Fig. Fig. Nine of 27 patients with undetected residual disease had tumour cells involving all layers of the oesophageal wall. Residual disease was present in the mucosa in 18 patients, and in the submucosa underneath a tumour‐free mucosa in eight patients. In one patient tumour cells were present underneath tumour‐free mucosal and submucosal layers Fig. 4b. In tTable 2). Of these, pathological material from endoscopic biopsies was available from 47 CREs. Specific mucosal tissue was detected in the biopsies from 34 of 47 CREs. Specific submucosal structures were identified in biopsies of three of 47 CREs from two patients, and the origin of the tissue was uncertain in ten of 47 CREs. No tumour cells were present in the biopsies that contained submucosal structures.The 27 patients underwent 54 CREs  ulceration, scarring and atrophy of the subepithelial layers of the oesophagus in several instances resulted in a more superficial location of these layers than expected. In this study, cancer cells were still located in the oesophageal mucosa in two‐thirds of patients with residual disease after nCRT that could not be detected by endoscopic biopsies during CREs. Furthermore, nearly one‐third of patients had undetected residual disease in the submucosa underneath a tumour‐free mucosa. Whether endoscopic biopsies or bite‐on‐bite biopsies had the potential to detect these submucosal tumours is unclear, as submucosal structures were identified in only two of the 27 patients with undetected residual disease. Only one patient had undetected residual disease in deeper layers of the oesophagus beneath a tumour‐free mucosa and submucosa.All patients included in the present study participated in a multicentre prospective trial with the objective to identify patients who might benefit from an active surveillance strategy in the future. As a result, all patients underwent standardized CREs at two fixed time points after completion of nCRT.3D®; CDx diagnostics, Suffern, New York, USA) that is able to sample larger areas of the oesophageal mucosal surface as deep as the muscularis mucosae. WATS has previously been used in an RCTUndetected residual disease was found in the mucosa in two‐thirds of patients, comparable to the findings of a previous retrospective studyversus 9 per cent in the study of Chao et al.10) as routine endoscopic biopsies do not have the potential to reach these deeper layers.Potentially valuable imaging or biomarker techniques include PET–CT with radiomics or circulating tumour DNA (ctDNA)In this study, 30 per cent of patients (8 of 27) had submucosal residual tumour below a tumour‐free mucosal layer, which is comparable to the 22 per cent reported previouslyFig. This study has several limitations. First, submucosal mucinous glands and thick‐walled vessels comprised only 1–2 per cent of the submucosal layer in the distal part of the non‐irradiated, normal oesophagus. Therefore, it cannot be concluded that the submucosa had not been sampled when these structures were absent from biopsies, especially if radiation‐induced atrophy and therefore the possible disappearance of these specific submucosal structures is also taken into consideration. Conversely, structures located in the deeper layers of the oesophageal wall in the healthy oesophagus could be present more superficially after nCRT owing to ulceration and fibrosis Fig. 5. As suTable S1 ypTNM status and TRG‐status of five patients with undetected residual disease of which resection specimens were not available for revision.Click here for additional data file.


Although novel teleconsultation solutions can deliver remote situations that are relatively similar to face‐to‐face interaction, remote assessment of heart rate and rhythm as well as risk factors remains challenging in patients with atrial fibrillation (AF).Hypothesis.Mobile health  solutions can support remote AF management.Herein, we discuss available mHealth tools and strategies on how to incorporate the remote assessment of heart rate, rhythm and risk factors to allow comprehensive AF management through teleconsultation.Particularly, in the light of the coronavirus disease 2019 (COVID‐19) pandemic, there is decreased capacity to see patients in the outpatient clinic and mHealth has become an important component of many AF outpatient clinics. Several validated mHealth solutions are available for remote heart rate and rhythm monitoring as well as for risk factor assessment. mHealth technologies can be used for (semi‐)continuous longitudinal monitoring or for short‐term on‐demand monitoring, dependent on the respective requirements and clinical scenarios. As a possible solution to improve remote AF care through teleconsultation, we introduce the on‐demand TeleCheck‐AF mHealth approach that allows remote app‐based assessment of heart rate and rhythm around teleconsultations, which has been developed and implemented during the COVID‐19 pandemic in Europe.Large scale international mHealth projects, such as TeleCheck‐AF, will provide insight into the additional value and potential limitations of mHealth strategies to remotely manage AF patients. Such mHealth infrastructures may be well suited within an integrated AF‐clinic, which may require redesign of practice and reform of health care systems. This may be particularly important for the management of patients who live remotely and far away from coordinating medical centers or during catastrophes, including coronavirus disease 2019 (COVID‐19), when attendance of outpatient clinics or traveling to the hospital is not possible or undesirable.3 AlthIn this review article, we discuss mobile health  tools and strategies to remotely monitor heart rate and rhythm and incorporate AF risk factors assessment to allow comprehensive AF management through teleconsultation. Additionally, as a possible solution to improve remote AF care during the COVID‐19 pandemic, we introduce the on‐demand TeleCheck‐AF mHealth approach that allows remote app‐based assessment of heart rate and rhythm around teleconsultations.2Different mHealth tools are available for remote heart rate and rhythm assessment. Until now, most tools are available within a patient‐initiated paying‐model which, together with the absence of reimbursement, complicates the clinical implementation and guidance of mHealth use by the treating physician. Additionally, the requirements on the way when and how to perform measurements , ECG‐documentation of an AF episode is required for diagnosis. As most PPG algorithms are developed for AF screening scenarios, they operate in a high sensitivity mode, resulting in a higher number of false positive recordings. Therefore, in the setting of AF screening, PPG‐detected episodes suggestive of AF need to be confirmed by an ECG. Despite crucial differences between PPG and ECG technologies for heart rhythm monitoring, most of the PPG‐based algorithms are validated to detect AF with a high sensitivity and specificity. Additionally, there is already some data that PPG technology is nearly as accurate as ECG to detect AF. In the validation studies for these devices, atrial high rate episodes (AHRE) from CIEDs have not been taken into account. Given the wide availability and low cost, PPG technology may represent an optimal screening tool to detect AF, which can then be confirmed by ECG technology afterwards in a second step.To screen for AF, (semi‐)continuous remote monitoring by cardiac implantable electronic devices (CIEDs), wearables , handheld devices  or app‐based mHealth solutions using PPG technology through the smartphone's built‐in camera  have been developed and validated Figure . Smartwa5, 6While PPG technology has limitations to diagnose AF, the wide accessibility and low cost of this technology via smartphone apps makes it an interesting tool for remote heart rate and rhythm monitoring of patients who have already been diagnosed with AF. A temporary pre‐determined on‐demand approach, where heart rate and rhythm information are provided to the health care provider just before a scheduled appointment appears to fulfill most requirements to manage AF patients remotely through teleconsultation. This on‐demand, rather than unfocused long‐term monitoring enables an ideal remote consultation with helpful and crucial well‐timed heart rate and rhythm information available for the treating physician, nurse or allied healthcare professional to steer management of patients with ECG‐documented AF. For an on‐demand mHealth approach, apps can be activated “on prescription” and linked to a secured cloud, which is accessible by the treating physician or nurse. Additionally, simultaneous monitoring of heart rhythm and symptoms provides information about symptom‐rhythm correlation. On‐prescription monitoring increases awareness with the patients who are better prepared for the remote consultation: patients know that symptom and rhythm evaluation is on the agenda during the consultation, together with discussions on necessary treatments. An on‐demand monitoring approach also avoids unnecessary data load, which would require work‐intensive and expensive data management infrastructures.3 it remains unclear, how best to assess risk factors and guide risk factor management and lifestyle modification in a remote setting. Established risk factors are often assessed only once in a structured way at the time point when AF patients present for the first time in the AF‐clinic (spot‐assessment of risk factors). However, several AF risk factors may show a high visit‐to‐visit or even day‐to‐day variability and lifestyle components such as physical activity, diet and sleep behaviors may be variable over time. This visit‐to‐visit or day‐to‐day variability does not just complicate the detection of AF risk factors but may also have a prognostic implication. High visit‐to‐visit variability in risk factors is associated with increased risk of incident new‐onset AF, worse cardiovascular outcome and increased mortality. Hence, assessment of risk factors requires a longitudinal and remote structured monitoring infrastructure  as well as the treatment team (smart technology to support decision making). In fact, such technology solutions should support integrated care in terms of actively involving patients in their care process, collaboration within multidisciplinary teams, and provide guidance in the complex (shared) treatment decisions and coordination of care.1) The  making).35 In involvement of the patient is promoted through the mHealth solutions. The role of the patient in an mHealth infrastructure is crucial, as the treatment team relies on the patient to use the infrastructure and collect and provide data on vital parameters such as heart rate and rhythm as well as symptoms and potential risk factors. However, before patients can take on such a task, they should be educated and clearly instructed, so they understand what is expected from them.2) Active multidisciplinary treatment team. This team consists of cardiologists, nurses, primary care physician and other specialists that might be involved in the management of AF, depending on the individual case. Infrastructure for such an approach might be available in terms of a specialized AF‐clinic, where cardiologists and specialized nurses would work closely with the patient, in a face to face setting or via teleconferencing, aiming to improve efficiency and outcomes. Communication is key in such teams to assure that all team members understand their role and contribution, and that there is a designated care coordinator. Depending on the context this may be a nurse within the AF‐clinic or administrative staff.3) The treatment may be provided by a F‐clinic,36 whedelivery of comprehensive treatment. Besides the management of AF  alone, it is crucial to determine the potential stroke risk and prescribe appropriate oral anticoagulation accordingly to prevent thromboembolic complications; management of precipitating factors (such as underlying cardiovascular conditions and modifiable risk factors) to reduce the cardiovascular burden to consequently reduce the AF burden.4) The final component of integrated care is the F burden.40These four fundamentals form the basis of an integrated AF care approach and the use of mHealth solutions through teleconsultations, seamlessly fits within an AF‐clinic as well as with the aims of integrated care: improving outcomes while preventing fragmentation of care. One possible pathway to implement an on‐demand mHealth infrastructure for remote heart rate, rhythm and risk factor assessment to allow comprehensive AF management through teleconsultation is shown in Figure 5). TeleCheck‐AF involves a structured teleconsultation (“Tele”) preceded by an app‐based on‐demand heart rate and rhythm monitoring infrastructure (“Check”) to guarantee comprehensive AF management (“AF”). The Conformité Européenne (CE)‐marked PPG‐based mobile phone app (www.fibricheck.com) allows semi‐continuous heart rate and rhythm monitoring of AF patients for 7 days prior to and during the teleconsultation. One important advantage of TeleCheck‐AF compared to other systems of telemonitoring is the on‐demand mHealth approach. It enables the physicians to use heart rate and rhythm data for treatment decisions and prevents unnecessary data collection which would be the case with continuous long‐term heart rate and rhythm telemonitoring systems , and which need to be managed afterwards requiring work‐intensive and expensive data management infrastructures. Additionally, the on‐demand heart rate and rhythm monitoring approach empowers patients to monitor their vital parameters and self‐manage their condition. Patients are involved in making decisions about measurement time and number of measurements during the day, that depends in particular on the presence of symptoms. Furthermore, the TeleCheck‐AF approach provides crucial information about symptom‐rhythm correlation by simultaneous rhythm and symptom assessment to steer appropriate AF management. The TeleCheck‐AF infrastructure can be combined with other available app‐based risk factor assessment tools, to allow the comprehensive remote assessment and management of AF patients.TeleCheck‐AF is an on‐demand mHealth intervention incorporating an app‐based heart rate and rhythm monitoring infrastructure to allow remote AF management through teleconsultation. During the COVID‐19 pandemic, it was made available in several European centres to keep AF patients out of the hospital .42 Tel6 An important element for embedding mHealth in clinical practice is the accessibility of the recordings by other healthcare professionals. For this, a connection with the patients' electronic healthcare record is crucial. This connection facilitates automatic transmission of the recordings from the secured cloud to the electronic healthcare record of the patient and increases the accessibility of the data for other healthcare professionals. Additionally, many AF apps lack scientific validation and are written at excessively high reading‐grade levels challenging users with limited health literacy. Although mHealth solutions for heart rate and rhythm monitoring are clinically established and used, apps for longitudinal risk factor assessment are not available and were therefore not yet incorporated in the TeleCheck‐AF approach. Finally, a multi‐disciplinary effort by regulatory agencies, healthcare organizations, and app stores is required to improve relevance, scientific validity, and readability of AF apps for AF patients. Additionally, discussions with insurance companies about reimbursement of mHealth infrastructures and with different stakeholders to agree on security and privacy regulations are initiated in different countries.Implementation of mHealth infrastructures require adaptation of existing care coordination and clinical pathways.43 An 7Health tools in the management of AF are becoming indispensable in current healthcare. Novel tools are able to remotely assess heart rate and rhythm and incorporate AF risk factor assessment to allow comprehensive AF management through teleconsultation. TeleCheck‐AF is one, but not the only possible solution to improve remote AF care during the COVID‐19 pandemic and will provide insight into the additional value and potential limitations of mHealth strategies to remotely manage AF patients. Such mHealth infrastructures may be well suited within an integrated AF‐clinic, which may require redesign of practice and reform of health care systems.The authors declare that there is no conflicts of interests.


However, the structural, energetic, and metabolic properties that achieve this balance, and hence optimize detoxication, are not well understood. Two features of detoxication enzymes that are frequently cited as contributions to promiscuity include the exploitation of highly reactive versatile cofactors, or cosubstrates, and a high degree of flexibility within the protein structure. This review examines these intuitive mechanisms in detail and clarifies the contributions of the classic ligand binding models ‘induced fit’ (IF) and ‘conformational selection’ (CS) to substrate promiscuity. The available literature data for drug metabolizing enzymes and transporters suggest that IF is exploited by these promiscuous detoxication enzymes, as it is with substrate‐specific enzymes, but the detoxication enzymes uniquely exploit ‘IFs’ to retain a wide range of substrates at their active sites. In contrast, whereas CS provides no catalytic advantage to substrate‐specific enzymes, promiscuous enzymes may uniquely exploit it to recruit a wide range of substrates. The combination of CS and IF, for recruitment and retention of substrates, can potentially optimize the promiscuity of drug metabolizing enzymes and drug transporters.Detoxication, or ‘drug‐metabolizing’, enzymes and drug transporters exhibit remarkable substrate promiscuity and catalytic promiscuity. In contrast to substrate‐specific enzymes that participate in defined metabolic pathways, individual detoxication enzymes must cope with substrates of vast structural diversity, including previously unencountered environmental toxins. Presumably, evolution selects for a balance of ‘adequate’  Drug metabolizing enzymes and drug transporters exhibit remarkable substrate promiscuity that is critical for their function as detoxication enzymes. These enzymes exploit mechanistic variations of substrate‐specific enzymes in order to achieve their promiscuity. For example, drug metabolizing enzymes utilize the same cosubstrates substrate specific enzymes, but they decouple substrate binding from cosubstrate activation. Drug metabolizing enzymes also exploit conformational flexibility in novel ways to enhance their promiscuity. AOaldehyde oxidaseCSconformational selectionCYPcytochrome P450EHepoxide hydrolaseFMOflavin monooxygenaseGSHglutathioneGSTglutathione transferaseH/DX MShydrogen/deuterium exchange mass spectrometryIFinduced fitmEHmicrosomal epoxide hydrolaseP‐gpP‐glycoproteinSULTsulfotransferaseUDPGAuridine 5'‐diphosphoglucuronic acidUGTuridine 5′‐diphospho‐glucuronosyl transferaseDetoxication enzymes and transporters play a critical role in drug metabolism and drug–drug interactions. In fact, some of these enzymes are colloquially referred to as 'drug‐metabolizing' enzymes and ‘drug transporters’, but it is useful to emphasize that they have evolved to play a much broader role in the detoxication of environmental compounds. Drug metabolism is the 'collateral damage' that is incurred by the remarkable promiscuity that these enzymes exhibit, which is critical for their detoxication function. Included among this cohort are cytochrome P450s (CYPs), UDP‐glucuronosyl transferases (UGTs), glutathione transferases (GSTs), flavin monooxygenases (FMOs), epoxide hydrolases (EHs), aldehyde oxidases (AOs), efflux transporters such as P‐glycoprotein (P‐gp), and others. Many of these enzymes exhibit both substrate promiscuity, which is the ability to bind and perform the same metabolic reaction on structurally unrelated substrates, and catalytic promiscuity, which is the ability to catalyze many types of chemical reactions on chemically distinct functional groups with significantly different local transition states as defined previously Arguably, detoxication enzymes are fundamentally different from 'normal' enzymes: Their functional value can be optimal when they exhibit only modest catalytic rate enhancement as long as it includes a wide range of substrates, including substrates that have been previously unencountered, rather than optimizing rates with any individual substrate. This difference demands consideration of the molecular properties that promote promiscuity. The properties that confer promiscuity to drug metabolizing enzymes are likely to be different from the properties that provide the optimal rate enhancements with any single substrate. However, this premise is clouded by our inadequate understanding of the relationship between specificity or promiscuity and rate enhancement. Examples exist for mutations that increase the specificity for the cognate substrate and for a general increase in flux for several substrates upon single mutation, where ‘flux’ is considered here as the rate of product formation at any specific concentration of substrate et al. have elegantly shown that some detoxication enzymes can be easily converted via directed evolution in vitro to equally promiscuous enzymes with orthogonal substrate promiscuity A great deal of attention has focused on the evolutionary importance of the other types of ‘promiscuity’, wherein substrate‐selective enzymes or their mutants have the ability to catalyze reactions with noncognate substrates kcat/KM values across a series of substrates and normalized to account for the structural diversity within the substrate series J‐values’ that are a relative measure of the ability of closely related enzymes to metabolize a range of substrates without preference for any specific one. The resulting scale of promiscuity J‐values ranges from 0 (perfect specificity for one substrate in the series) to 1 (no preference for any substrate over another within the series). Drug metabolizing enzymes have J‐values > 0.7, whereas their corresponding substrate‐specific homologs have J‐values between 0.3 and 0.6 J‐values of ~ 0.8 and near 0, respectively, in accordance with their physiological functions The suggestion that detoxication enzymes are quantitatively more promiscuous than their structurally related substrate‐specific homologs is supported by application of a quantitative index in a few cases, based on relative kcat or kcat/KM, or ‘flux’ of substrate to product. For example, classic work of Knowles & Albery, and others, describes the evolutionary perfection of enzymes that starts with ‘uniform binding’ or equivalent stabilization of substrate complexes, product complexes, and transition states kcat/KM as criteria to be optimized et al., further evolution leads to ‘differential’ stabilization of the rate‐limiting transition‐state vs. ground‐state substrate or product complexes. This energetic perspective suggests that substrate‐specific enzymes perfect catalysis by avoiding clear rate‐limiting steps and having nearly equal energy barriers when many steps are involved In order to consider the possible attributes of an enzyme that optimizes promiscuity, it is useful to consider first some properties that contribute to substrate specificity, which are well established and understood. Benchmarks for the limits of substrate specificity and catalytic perfection are rooted in structural, kinetic, and energetic considerations. Energetic and kinetic criteria for optimization of substrate‐specific enzymes are based on kcat or kcat/KM, with the specific cognate substrate(s), without considering the interactions with noncognate substrates. These ideas have been amplified and refined by others in the context of promiscuous enzyme templates Notably, all of these mutational processes that lead to catalytic perfection during evolution are assumed to affect interactions with the cognate substrate on which the enzyme normally acts. It is presumed in analyses of evolutionary processes that the optimal changes in energetic profiles would be those that improve catalysis, either Structural considerations also reveal mechanisms of substrate specificity. In fact, it might be argued that the structural biology revolution demystified the ‘amazing’ substrate specificity attributed to many enzymes in the infancy of enzymology. As a result of the structural biology revolution of the 1980s–1990s, our understanding of enzyme 'specificity' is reasonably mature. We have learned how enzymes from many structural families can recognize specific substrates with great selectivity compared to close structural substrate analogs, by straightforward exploitation of shape, charge, hydrophobic interactions, and hydrogen bonding complementarity In contrast to the devices used by substrate‐specific enzymes, we understand little about the mechanisms by which drug metabolizing enzymes optimize their function. How does evolution optimize promiscuous enzymes in the absence of a defined substrate to define selective pressure? Arguably, it is a more challenging problem to evolve or design an enzyme that efficiently metabolizes many structurally diverse substrates than to design or evolve a highly specific enzyme that only needs to perform a single reaction. Flux of any individual substrate to product is unlikely to be the most important catalytic property for promiscuous detoxification enzymes, so it is difficult to superimpose models for the optimization of substrate‐specific enzymes on detoxication enzymes. Still, an interesting analysis of the detoxication aldose reductase in terms of flux and kinetic parameters has suggested the importance of cofactor binding over substrate binding to one‐way flux pKa of the thiol of bound glutathione (GSH) to ~ 6.7 so that GSH is largely ionized as the nucleophilic thiolate, GS‐, at the active site ‐ relative to the protonated thiol when GSH is bound, via hydrogen bonding to an active tyrosine or serine pKa of bound GSH of ~ 6.8 pKa of > 9, and is not poised to react with 'any nearby electrophile'. Presumably, long‐chain lipid aldehydes specifically induce deprotonation of GSH, as suggested by Hubatsch and Mannervik, although this has not been directly demonstrated Three decades ago, Bill Jakoby noted in review articles that the promiscuous detoxication FMOs, sulfotransferases (SULTs), and GSTs stabilize the reactive form of their respective cofactors or cosubstrates in the absence of any substrate pKa of GSH to generate more GS‐ and decreasing the nucleophilicity of the resulting thiolate as for their bound GSH and this could contribute to the collective substrate promiscuity of GSTs as of these GSTs near the maximum predicted by Bronsted behavior for a range of electrophilic substrates.An interesting extension of the reactive cofactor role in substrate promiscuity is the possibility that multiple isoforms of an enzyme with slightly different chemical reactivities of their cofactor could collectively achieve greater promiscuity and afford better protection from the widest range of toxins. This theoretical possibility has been considered with GSTs The CYPs dominate drug metabolism and provide an impressive level of protection from environmental toxins In fact, in many cases several catalytic reactions are performed by a single CYP on the same substrate. A drug or toxin with multiple functional groups or sterically unhindered carbon atoms may yield a heterogeneous mixture of metabolites from a single CYP isoform. This ‘product promiscuity’ is an additional affirmation of Jakoby’s initial mechanistic insight; detoxication enzymes generate highly reactive cofactors that wait for a substrate to ‘bump into them’, without orchestrating a specific orientation for a transition state. The wealth of intramolecular kinetic isotope effects that lead to metabolic switching (isotope‐dependent change in regioselectivity) is observed with CYP reactions, and the distinct lack of regioselectivity when multiple oxidizable sites are present within a single substrate is consistent with highly dynamic substrate complexes that allow the substrate to sample multiple orientations and allow multiple possible sites of oxidation to bump into the reactive cofactor max for FMOs is independent of the substrate being oxygenated, which presumably prompted Jakoby and others to consider the difference between detoxication enzymes and substrate‐specific enzymes. This substrate independence of rate is paralleled by substrate‐independent formation of the 4a‐hydroperoxy flavin intermediate that reacts with nucleophilic substrates. A distinctly different group of FMOs from prokaryotes, known as Baeyer–Villiger FMOs, uses the 4a‐hydroperoxy flavin intermediate as a nucleophile, wherein the distal oxygen attacks a carbonyl. Apparently, evolution has repurposed this intermediate for various functions, including oxidation of nucleophilic xenobiotics with the drug‐metabolizing FMOs As noted above, FMOs were among the enzymes considered by Jakoby and they contribute to the oxidation of drugs with nucleophilic heteroatoms including tertiary amines The hepatic UGTs, individually and collectively, also metabolize an extraordinary range of structurally unrelated drugs The cytosolic hepatic SULTs catalyze the sulfation of a wide range of xenobiotic phenols, alcohols, and amines Aldehyde oxidase performs a wide range of chemical oxidations on structurally diverse substrate aldehydes and aza‐ or oxo‐heterocycles and has recently become appreciated for its contribution to drug metabolism In addition to enzymes that perform metabolic transformations, many efflux transporters contribute to detoxication of environmental toxins and drugs by pumping them out of cells and facilitating their clearance The examples summarized above indicate the relevance of Jakoby’s early suggestion that highly reactive cofactors are a common device used by promiscuous enzymes. As suggested by Jakoby, recent studies based on systematic and extensive mutagenesis studies with mutants of a bacterial detoxification FMO lead to the conclusions that the role of the protein is only to provide a ‘shell’ for the reactive cofactor, rather than to contribute to interactions with substrates Despite these examples, the presence of reactive cofactors among detoxication enzymes is not a strictly necessary condition for substrate promiscuity. For example, some microsomal EHs (mEHs) considered to be detoxication enzymes do not use cofactors. The mEHs utilize an active‐site aspartic acid carboxylate to attack the epoxide of a drug or toxin and generate a covalent acylated substrate that is hydrolyzed to regenerate enzyme and free substrate diol Conversely, many highly substrate‐specific enzymes utilize the same cofactors and cosubstrates as those mentioned above. So, the presence of these cofactors or cosubstrates is not a sufficient condition for substrate promiscuity . In summary, despite their prevalence among detoxication enzymes, reactive cofactors or cosubstrates are neither a necessary nor a sufficient condition for substrate promiscuity.In addition to the exploitation of reactive cofactors, Jakoby suggested that a second property of detoxication enzymes that contributes to promiscuity is a conformationally flexible active site that can accommodate a wide range of substrates in proximity to the cofactors or cosubstrates IF refers to the case when a single ‘inactive’ enzyme conformation in the absence of substrate binds to the substrate, which induces a change to an active conformation. This ubiquitous behavior among enzymes provides a source of substrate specificity, as long as only the cognate substrate induces the correct conformational change In contrast, conformational selection occurs when an equilibrium of conformations exists in the absence of substrate, which selects one ‘active’ conformation to bind to. For substrate‐specific enzymes, CS has no advantage, and equilibrating states in the absence of ligand do not provide any catalytic or specificity advantage. An equilibrium of states in the absence of substrate, as in CS, is the cost that is paid to maintain the flexibility required for IF, because the nonbinding conformations of the substrate‐free ensemble could interact with noncognate substrates and lead to inhibition, or these conformations would decrease the apparent affinity of substrate for the active conformation. For conformational selection, the conformational change occurs before binding and cannot be advantageous for flux of cognate substrate to product.It is likely that the vast majority of enzymes exhibit combinations of both behaviors, which are not mutually exclusive. In fact, IF and CS are thermodynamically equivalent, path‐dependent, extremes of a continuum and it may be unrealistic to assign ‘one or the other’ to any individual enzyme. To the extent that these behaviors require flexibility or plasticity, IF and CS are natural starting points for considering the widespread expectation that flexibility correlates with promiscuity.J‐values for GSTA1‐1 and GSTA4‐4 are 0.79 and 0.43, respectively As noted above, two GST isoforms represent benchmarks for high specificity and high promiscuity within a single protein fold and they provide insight into a thermodynamic and kinetic base for promiscuity, which can be interpreted in the context of IF vs. CS. The GST isoform GSTA1‐1 is highly promiscuous, whereas GSTA4‐4 has evolved specifically to clear 4‐hydroxynonenal or long‐chain lipid aldehydes J‐values. One mutant, called the GIMFhelix, has ~ 600‐fold greater activity toward HNE than GSTA1‐1 does, and its crystal structure reveals a highly ordered immobile C‐terminal helix An obvious difference in protein dynamics between these isoforms lies at their C termini, which contribute to their active sites. The C‐terminal 14 residues of GSTA1‐1 form a mobile helix in the absence of substrates, with low electron density in crystallographic models and poorly resolved NMR peaks  revealed a low‐temperature reversible transition, prior to the main unfolding, with GSTA1‐1 that is absent with GSTA4‐4. This DSC transition was assigned to temperature‐dependent conformational flexibility of the C terminus of GSTA1‐1, based on several criteria J‐values were determined. The results demonstrate that, across the series of wild‐type and GST mutants, the width of the enthalpy landscape increased and the height of barriers between enthalpy states decreased, as the J‐values increased. The results with these GSTs indicate that conformational flexibility, as measured by these thermodynamic parameters, correlates with substrate promiscuity within this single protein fold The catalytic activity of each of the mutants was also determined with a range of substrates that represented a significant extent of chemical space, and their promiscuity Kinetic properties of these GSTs further revealed mechanistic aspects of their promiscuity. Pre‐steady kinetic analysis of the metabolism of two different substrates with the two most promiscuous GST enzymes in the series indicated that a conformational relaxation of the substrate‐free enzyme is kinetically significant for some, but not all substrates. That is, different combinations of substrate and GST variant differentially yielded pre‐steady‐state kinetic lags or bursts kcat/KM for any substrate that induces a fit via retention of bound substrate in the active site. Notably, this suggested behavior is supported by results with a nonenzymatic antibody. Antibodies are usually considered to be highly specific binders of their antigens, and they do not approach the degree of promiscuity exhibited by detoxication enzymes. However, behavior analogous to the proposed utility of CS in detoxication enzymes has been demonstrated by Tawfik et al., wherein a conformational ensemble of an antibody in the absence of antigen includes conformations that prefer a noncognate antigen, and this contributes to binding promiscuity The results are consistent with a model wherein these promiscuous GSTs sample many conformations in the absence of substrate, and different substrates select different conformations. This presentation of multiple conformations with different ‘substrate selectivities’ increases the promiscuity. Upon substrate binding, the flexibility of the proteins is sufficient to allow each substrate to induce the fit required to form a productive catalytically active complex. This model is schematized and compared to IF and CS for substrate‐specific enzyme in Fig. These examples support the possible contribution of CS and IF to the complex kinetics observed with GSTA1‐1, and they indicate the role of CS in increasing the level of promiscuity. The interesting extension of these results is that detoxication enzymes have optimized these behaviors to further increase their substrate promiscuities.3, 1650 Å3, and 2000 Å3, respectively Support for the contribution of the noncanonical type of IF to substrate promiscuity of drug metabolizing enzymes comes from numerous crystal structures, wherein different substrates bound to the same detoxication enzyme induce significantly different protein conformations. For example, CYP3A4 is the most promiscuous CYP isoform and different drugs or ligands induce structural changes that are widely distributed throughout the entire protein and different structural elements adopt ligand‐dependent locations and conformations Recent kinetic data indicate that several promiscuous drug‐metabolizing CYPs and some substrate‐specific CYPs utilize CF Regarding the potential contribution of CS to promiscuity, P‐gp presents a striking example of conformational heterogeneity in the substrate‐free enzyme. A few crystal structures of human and mouse P‐gp are available with drugs or substrates bound, so structural evidence for a wide range of substrate‐dependent conformation exists Importantly, the conformational heterogeneity of the substrate‐free P‐gp is distinctly different from GSTA1‐1, in as much as the energy landscape must include significant energy barriers to rearrangement, in contrast to the barrierless, fluid, and landscape of GSTA1‐1. Speculatively, if substrates for P‐gp do not drive the protein to a homogeneous ensemble on timescales similar to transport, then only a portion of the enzyme population would participate in the transport at early times during catalytic transport. With extended exposure to the same substrate, the entire P‐gp population might be engaged, as suggested by model studies for generic enzymes exhibiting slow conformational changes.Drug metabolizing enzymes are among the most substrate and catalytically promiscuous of any enzymes, with properties that contrast some of the properties of promiscuous enzymes that are formed from random mutations of substrate‐specific enzymes, as schematized in Fig. The authors declare no conflict of interest.


Significant efforts are necessary to introduce new dietary protein sources to feed a growing world population while maintaining food supply chain sustainability. Such a sustainable protein transition includes the use of highly modified proteins from side streams or the introduction of new protein sources that may lead to increased clinically relevant allergic sensitization. With food allergy being a major health problem of increasing concern, understanding the potential allergenicity of new or modified proteins is crucial to ensure public health protection. The best predictive risk assessment methods currently relied on are in vivo models, making the choice of endpoint parameters a key element in evaluating the sensitizing capacity of novel proteins. Here, we provide a comprehensive overview of the most frequently used in vivo and ex vivo endpoints in murine food allergy models, addressing their strengths and limitations for assessing sensitization risks. For optimal laboratory‐to‐laboratory reproducibility and reliable use of predictive tests for protein risk assessment, it is important that researchers maintain and apply the same relevant parameters and procedures. Thus, there is an urgent need for a consensus on key food allergy parameters to be applied in future food allergy research in synergy between both knowledge institutes and clinicians. AgallergenALAalpha‐lactalbuminBATbasophil activation testBLGbeta‐lactoglobulinBNBrown NorwayDCdendritic celle.c.epicutaneouslyELISAenzyme‐linked immunosorbent assayGM‐CSFgranulocyte‐macrophage colony‐stimulating factori.c.intracutaneousi.d.intradermallyi.g.intragastrici.n.intranasali.p.intraperitoneallyIgimmunoglobulinILCinnate lymphoid celli.v.intravenousKSCNpotassium thiocyanateNAnot analysedNKTnatural killer T cellOVAovalbuminPCRpolymerase chain reactionRBLrat basophil leukemias.c.subcutaneouslySPTskin prick testTregregulatory T cellTSLPthymic stromal lymphopoietinWPCwhey protein concentrateWPHwhey protein hydrolysate1A variety of in vitro and in vivo models have been developed that address the factors and mechanisms involved in the sensitization to food proteins.2In murine‐type models of food allergy to milk, eggs, and peanuts, a drop in the core body temperature is often observed after repetitive allergen challenge. This change in body temperature is an indicator of anaphylaxis Table . Tempera2.1The measurement of core body temperature is a cost‐effective, reliable assessment of the allergic reaction.Therapeutic or preventative strategies for the reduction of allergic reactions can be easily evaluated.Can be used to evaluate the severity of allergic shock and differences between allergens subjected to physical transformations .2.2The occurrence of anaphylaxis is dependent on the mouse strain used: Balbc or C3H mice are prone to develop anaphylaxis, whereas C57BL/6 or A/J mice necessitate stringent exposure protocols to achieve sensitization.The clinical score may be biased as a consequence of the laboratory environment, stress level, animal strain, and technical experimenter.A decrease in temperature is only observed after a food/allergen challenge after a previous sensitization event; this endpoint therefore contains no predictive value for the sensitization potential of a food protein.2.3Using a rectal probe, mice or rats must be acclimated to the experimental room at least 1 hour before starting the temperature measurements to obtain stable values.The rectal temperature must be evaluated 10 minutes to 1.5 hours after the challenge.The animal temperature can be registered over time using a programmable temperature transponder implanted subcutaneously.3While in vivo measurements are essential to assess the elicitation of an allergic response, they do not provide insight into de novo allergen sensitization. Therefore, blood, tissue, or organs must be collected and further analyzed by ex vivo methods. Serum immunoglobulin (Ig) content is the most common parameter measured when evaluating sensitization to food allergens in animal models, followed by fecal IgA see Data , as anti3.1Specific IgE antibody analysis is the most trustworthy measure of sensitization.Measures of specific IgE antibodies are often used to evaluate not only sensitization but also the potential severity of the allergic reaction after a second encounter.Measurements of antibodies can be performed without the use of advanced equipment such as a cytometer or robotics.3.2Assays often need to be developed in‐house, restricting the possibilities for comparison between laboratories.IgE only accounts for a fraction of all serum antibodies, requiring more advanced ELISAs for analysis of specific IgE.IgE levels do not predict the clinical severity of a food allergy model, and other ex vitro experiments are needed to further address this parameter.Measures with optical density (OD) as the unit only allow for one serum dilution.3.3Antibody‐capture ELISAs should be used for the measurement of specific IgE.Other antibody parameters in addition to the amount of total and specific antibodies are relevant and should be measured, such as clonality and avidity.Measures of total and specific antibodies should always be expressed as titer values or as concentrations with no upper or lower limit for dilutions.Serum depleted of IgG using protein G columns before use in indirect ELISAs needs to be considered.4+ T cells in mesenteric lymph nodes, the spleen, and Peyer's patches.+ T cells derived from mesenteric lymph nodes of OVA‐sensitized mice is sufficient to transfer allergen‐induced diarrhea to naïve recipients. The recipient mice also display an upregulation of the Th2‐related chemokines CCL17 and CCL22 in the small intestine.+ Foxp3+ T cells associated with tolerance are often compromised in many food allergy models.Assessment of serum Ig levels provides important information about the sensitization phase but does not allow for quantification of immune cell responses, including cellular infiltration to sites of allergic inflammation. The phenotyping of innate  and adaptive (B and T cells) responses is indispensable for assessing the mechanisms of allergic sensitization Table . Immune 4.1Precise mechanistic insights into the cellular response in isolated organs and tissues support the sensitizing potential of food proteins when combined with additional readouts.Precise determination of the T‐cell profile by using specific markers of the T‐cell population.Quantitative evaluation of the infiltrating cell population by flow cytometry.4.2Analysis of cell populations without the contribution of neighboring cell tissue (loss of microenvironment).Isolation of immune cells from tissues relies on enzymatic digestion protocols and may thus alter phenotypical and functional properties of the cells of interest.Difficulty with the separation of minor subpopulations.Sacrifice of the animal is required for organ and tissue sampling.Need for sophisticated equipment such as FACS.Type 2 immune response‐associated mucus production in tissues makes cell isolation difficult and can create bias in cell phenotyping and frequencies.4.3Remove fat and store organs, tissues and cells at 4°C to avoid uncontrolled cell death or degradation of surface markers.Perform flow cytometry and culturing the same day as the animal kill.Phenotyping of T cells can be achieved by intracellular cytokine/transcription factor staining using flow cytometry.5The logical follow‐up to the analysis of infiltration/expansion of innate and adaptive immune cells in the tissues and organs is the evaluation of cytokine secretion. This evaluation comes directly from serum or from lymphatic tissue cells restimulated ex vivo. Food allergen stimulation of only lymphatic tissue cells, or in coculture with dendritic cells, allows for the immunophenotyping of the immune cell populations specific for the exposed food antigen or matrix. To confirm allergen specificity, splenocytes, mesenteric lymph node cells, or lamina propria cells isolated from sensitized and/or challenged mice are restimulated with corresponding allergenic proteins or peptides. After culture for up to 5 days, cytokines associated with the inflammatory response  and the regulatory response (IL‐10 and TGF‐β) are analyzed in the supernatants by ELISA5.1Precise assessment of the allergen specificity by restimulating cells with the same allergen used in the animal model.Class determination of the T‐cell response by evaluation of cytokine production in the supernatant of sorted T cells.Higher production of cytokines can be obtained after proliferation and restimulation with the antigen than by direct measurement in serum.5.2Restimulation with allergens can activate nonspecific T cells due to certain cross‐reactivity.Difficult to obtain a level above the sensitivity threshold with cells isolated from naïve mice.Some mechanistic endpoints are not equally important in animals and humans.5.3For allergen presentation, presorted T cells need to be co‐cultured with dendritic cells.MHC peptide—tetramers can be used to sort specific T cells and have better assessment of allergen specificity.Need for positive  and negative control  stimuli to ensure proper T‐cell responsiveness.Endotoxin levels within the allergen extract need to be controlled to prevent bias in restimulation responses.Ideally, when using gene expression sequencing data, this method should be confirmed with at least one other technology .As cells and mediators associated with immune responses change rapidly, longitudinal assessments of mechanistic endpoints will be more informative than single time point assessments. The timing of the measurements will depend on the research question, for example, sensitization mechanisms vs mechanisms of acute allergic responses following (re)challenge.6To date, the methods to study intestinal pathophysiology are in vitro culture systems with cell lines or explanted mucosa grown in monolayers,6.1The tissue structure, cellular components, and neural system are highly preserved.The model provides the possibility to study immediate responses generated after the introduction of different molecules and microbes.6.2Only short‐term responses can be evaluated due to changes that can occur in the tissue over time.Currently, only intestinal segments from 12‐ to 14‐day‐old mice have been tested.Tissue preparation and assembly require specific skills.7The recent broadening of our knowledge of food allergy pathogenesis and development of murine food allergy models has enabled us to model the allergic elicitation reaction as well as the preceding sensitization events and observe relevant symptoms with different food proteins . The principal endpoint parameters described in this review are critical parameters that should be evaluated in a correct manner so that they may be powerful in the different rodent models.Characterizing a food allergy model using temperature, level of Igs, phenotyping of the cell infiltrate, and cytokine production gives an overview of the reaction while providing us insight into the degree of sensitizing capacity of the allergen used. Nevertheless, even though the in vivo measurements and the ex vivo experiments provide us with many answers about the immune response and the sensitization phase, we still do not have a complete overview of the immune mechanisms behind each reaction. There is still a strong need to better define the allergic reaction to predict the clinical outcomes of sensitization to novel food proteins. Although the current available models are suitable for studying the pathophysiology of food allergy, they still cannot predict the magnitude of the allergic potential of a particular allergen. Discovering and highlighting the molecules and cells involved in both sensitization and elicitation are necessary to improve risk assessment models and to facilitate the introduction of novel protein sources into our diet with a low risk of allergic sensitization.Dr Blanchard reports other from Nestec, outside the submitted work; Dr O'Mahony reports personal fees from AHL and grants from GSK, outside the submitted work. The other authors declare no conflict of interest. Click here for additional data file.


As part of the Single Technology Appraisal process, the National Institute for Health and Care Excellence (NICE) invited Pierre Fabre to submit evidence for the clinical and cost-effectiveness of encorafenib with binimetinib (Enco + Bini) versus dabrafenib with trametinib (Dab + Tram) as a first-line treatment for advanced (unresectable or metastatic) BRAF V600 mutation-positive melanoma. The Liverpool Reviews and Implementation Group at the University of Liverpool was commissioned as the Evidence Review Group (ERG). This article summarises the ERG’s review of the company’s evidence submission (CS), and the Appraisal Committee’s (AC’s) final decision. The main clinical evidence in the CS was derived from the COLUMBUS trial and focused on the efficacy of Enco + Bini (encorafenib 450 mg per day plus binimetinib 45 mg twice daily) compared to vemurafenib. The company conducted network meta-analyses (NMAs) to indirectly estimate the relative effects of progression-free survival (PFS), overall survival (OS), adverse events (AEs) and health-related quality of life (HRQoL) for Enco + Bini versus Dab + Tram. None of the results from the NMAs demonstrated a statistically significant difference between the treatment regimens for any outcomes. The ERG advised caution when interpreting the results from the company’s NMAs due to limitations relating to the methods. The ERG considered that use of the OS and PFS hazard ratios (HRs) generated by the company’s NMAs to model the relative effectiveness of Enco + Bini versus Dab + Tram in the company model was inappropriate as these estimates were not statistically significantly different. The ERG amended the company’s economic model to include estimates of equivalent efficacy, safety and HRQoL for Enco + Bini and Dab + Tram. The ERG considered use of different estimates of relative dose intensity to be inappropriate and used the same estimate for both drug combinations. The ERG also concluded that as only the prices of drug combinations were different, a cost comparison was an appropriate method of economic analysis. Using this approach , treatment with Enco + Bini was more cost effective than treatment with Dab + Tram. The AC raised concerns that an absence of evidence of a difference in outcomes between Enco + Bini and Dab + Tram did not constitute evidence of absence. However, as the numerical differences in outcomes generated by the company’s networks were small, the AC did not have a preferred approach and considered that both the company’s and the ERG’s methods of incorporating outcome estimates into the economic model were suitable for decision making. The NICE AC recommended Enco + Bini as a first-line treatment for unresectable or metastatic melanoma with a BRAF V600 mutation. The National Institute for Health and Care Excellence (NICE) is an independent organisation responsible for providing national guidance to the National Health Service (NHS) in England and Wales on a range of clinical and public health issues, including the appraisal of new health technologies. The NICE Single Technology Appraisal (STA) process is specifically designed for the appraisal of a single health technology for a single indication, where most of the relevant evidence lies with one manufacturer or sponsor and typically covers new technologies shortly after UK market authorisation is granted . Within Using a specification developed by NICE , the NICE Appraisal Committee (AC) considers the company’s submission , the ERGThis article presents a summary of the ERG report for the STA of encorafenib with binimetinib (Enco + Bini) as a treatment for patients with advanced (unresectable or metastatic) BRAF V600 mutation-positive melanoma. The Liverpool Reviews and Implementation Group at the University of Liverpool was commissioned to act as the ERG for this STA. Full details of all relevant appraisal documents  can be found on the NICE website .Melanoma is the fifth most common cancer in the UK . IncidenIn the UK, in 2016, almost 14,000 new cases of melanoma were diagnosed . ApproxiAlmost half of all melanomas express a mutated form of the B-Raf proto-oncogene kinase (BRAF) and the majority of these are BRAF V600 mutations . The speEncorafenib is licensed in Europe  for use NICE developed a scope for the assessment of Enco + Bini, which specified that the clinical and cost-effectiveness of this combination treatment should be established within its licensed indication relative to Dab + Tram. Five measures of clinical effectiveness were considered relevant for this appraisal: overall survival (OS), progression-free survival (PFS), response rate, adverse events (AEs) and health-related quality of life (HRQoL). If sufficient evidence was available, then subgroup data was to be considered for people with previously untreated disease and for people with previously treated disease that had progressed on or after first-line immunotherapy.To assess whether the company’s submission conformed to the methodological guidelines issued by NICETo assess whether the company’s interpretation and analysis of the evidence were appropriateTo indicate the presence of other sources of evidence or alternative interpretations of the evidence that could help inform NICE guidance.The company provided a submission to NICE describing the use of Enco + Bini (within the context of its licensed indication) in adults with advanced (unresectable or metastatic) BRAF V600 mutation-positive melanoma. The comparator for this appraisal was Dab + Tram . The ERGIn addition to providing this detailed critique, the ERG modified a number of key assumptions and parameters within the company’s economic model to examine the impact of such changes. This section summarises the evidence submitted by the company and the ERG’s review of that evidence.The company conducted a literature search that identified a single randomised controlled trial (RCT), the COLUMBUS trial –23. The p < 0.0001.The direct evidence from the COLUMBUS trial –23 is prp values for OS from the interim OS analysis (7 November 2017) were, therefore, only descriptive. Median OS was 33.6 months (95% CI 24.4–39.2) in the Enco + Bini arm and 16.9 months (95% CI 14.0–24.5) in the vemurafenib arm. The HR for the comparison of Enco + Bini with vemurafenib was 0.61 . Results of updated, supportive and sensitivity analyses of primary and key secondary efficacy outcomes were consistent with the results of the primary analysis.OS outcomes could not be formally tested, according to the pre-specified hierarchical approach of statistical testing, since Enco + Bini did not demonstrate a statistically significant advantage over Enco . Nominal The HRQoL results from the COLUMBUS trial –23 demonThe frequencies of AEs were similar across the three arms of the COLUMBUS trial –23. PatiThe ERG noted that the clinical efficacy outcomes and the HRQoL outcomes of the COLUMBUS trial –23 favouIn the absence of direct evidence comparing Enco + Bini versus Dab + Tram, the company conducted Bayesian network meta-analyses (NMAs) to indirectly estimate the relative effects of treatment efficacy (PFS and OS), safety and HRQoL. The company identified seven RCTs , while results for serious AEs favoured Enco + Bini (OR < 1). However, for both analyses, the CrIs crossed 1, meaning a statistically significant difference was not demonstrated.The ERG was satisfied with the company’s literature search strategy approach and was confident that the search was carried out to an acceptable standard. The ERG did not identify any additional studies that should have been included in the company’s review.The ERG considered that the COLUMBUS trial –23 was oThe results from the COLUMBUS trial –23 did nThe ERG interpreted the results of the company’s NMAs with caution due to several issues relating to the methods, including the sparsity of evidence in the networks (particularly the HRQoL network), the variability in the lengths of trial follow-up (2–6 years), differences between trials in median follow-up for OS 11–33.3 months), the inclusion of dacarbazine within the networks (which is not a BRAFi and is no longer regarded as a standard NHS treatment), and the fact that only an NMA of PFS by local investigator review (rather than BIRC) was feasible. Five of the seven trials  BRAF V600 mutation-positive melanoma. Using Microsoft Excel, the company produced a partitioned survival model with a 30-year time horizon and monthly cycles. The UK NHS was the perspective of the model, in line with the NICE reference case . OutcomeConfidential Patient Access Scheme (PAS) prices, discounted prices for the NHS, agreed with the Department of Health, were in place for Enco + Bini and for Dab + Tram at the time of this appraisal. As the company was unaware of the PAS prices for the comparator treatments, the company base-case results only included the PAS price for Enco + Bini. Using this reduced price, the results from the company’s economic model estimate that Enco + Bini dominates Dab + Tram, generating 0.453 additional QALYs at a reduced cost.The company carried out probabilistic and deterministic sensitivity analyses. The results showed that the base-case analyses results were sensitive to the use of an estimated HR for time to treatment discontinuation (TTD)  and the dose of Dab + Tram (drug dose each time it was administered and RDI). Two scenarios in which Enco + Bini did not dominate Dab + Tram were produced by the company; one of which included discounts to the list prices of dabrafenib and trametinib, and the other assumed Enco + Bini and Dab + Tram were equally effective and safe .The ERG considered that the design of the company model was appropriate and that it was generally well structured. In addition, the ERG was satisfied with the way in which the COLUMBUS trial –23 data The COLUMBUS trial –23 data Based on the lack of any evidence supporting a demonstrable difference in outcomes between Enco + Bini and Dab + Tram, the ERG considered that the only parameters that could affect model results were treatment-related costs. In the company model, these were a function of time on treatment, administration costs, RDI and drug costs. The ERG was satisfied that the time on treatment estimates and the administration costs (given the same mode of delivery) for Enco + Bini and Dab + Tram were the same, as assumed in the company base case. The company employed different RDI multipliers in their estimation of treatment costs for Enco + Bini and Dab + Tram. However, as the ERG considered that there was no robust evidence to support differential AE estimates for each of the treatment options, there was equally no robust evidence to support the use of different RDI multipliers.The ERG’s opinion was that equivalence of time on treatment, drug administration costs and RDI across treatment options meant that drug prices were the only cost difference to consider; therefore, to establish cost-effectiveness, a simple cost-minimisation analysis, rather than a cost-utility analysis, was appropriate.The objective of this appraisal was to compare the clinical and cost-effectiveness of Enco + Bini versus Dab + Tram for adults with advanced (unresectable or metastatic) BRAF V600 mutation-positive melanoma. As Dab + Tram was not a comparator in the COLUMBUS trial –23, the The lack of direct comparative evidence between Enco + Bini and Dab + Tram resulted in the company carrying out NMAs to provide estimates for each of the key outcomes. The networks did not, however, generate estimates of effects for Enco + Bini that statistically significantly differed from estimates for Dab + Tram. The ERG considered that estimating differences between Enco + Bini versus Dab + Tram based on estimates from the NMAs in the economic model was inappropriate and, therefore, carried out a cost-minimisation analysis.As the company assumed time on treatment to be the same for Enco + Bini and Dab + Tram, and in the ERG’s scenarios the key outcomes  were assumed to be the same for both treatment options, the ERG considered that the proposed difference in RDI in the company model was difficult to justify. The ERG adjusted the model so that the RDI was the same for the two comparator treatments.The AC heard from a patient and clinical experts that having the choice of two targeted combination treatments would improve patients’ HRQoL, particularly given that Enco + Bini and Dab + Tram have different toxicity profiles. The AC concluded that both clinicians and patients would welcome an additional treatment option.The AC heard from clinical experts that the treatment of patients with unresectable or metastatic BRAF V600 mutation-positive melanoma is evolving, with new immunotherapy and other treatments becoming available. There is also variation in practice across the NHS as to the use of targeted therapies or immunotherapies, alone or in combination, as first-line therapy; however, clinical experts agree that most patients will receive targeted therapies at some time. The clinical experts discussed a preference for the use of immunotherapies in the first-line setting for patients with a good PS, but stated that this was not a practice adopted throughout the NHS. The AC noted that the position of targeted therapy in the treatment pathway was not easy to establish.The AC heard from the ERG that the population in the COLUMBUS trial –23 had aAlthough the ERG raised a potential concern that the proportion of patients receiving immunotherapies as a first-line treatment in the COLUMBUS trial –23 was lThe AC considered the COLUMBUS trial –23 to beThe AC noted the lack of statistically significant differences in PFS, OS, AEs or HRQoL demonstrated in the company’s NMAs. The AC considered that the results of the NMAs should be viewed with caution due to their wide CrIs in all base-case and sensitivity analyses and the limitations highlighted by the company and ERG in terms of the construction of the networks. The clinical experts indicated that Enco + Bini and Dab + Tram are likely to have similar clinical effectiveness estimates. The AC concluded that clinical effectiveness estimates are likely to be similar, but are associated with uncertainty.The AC considered the rates of AEs within the COLUMBUS trial –23 to beThe AC was satisfied that the company’s model was appropriately structured for decision-making. The AC accepted the ERG’s amendment to RDI to Enco + Bini, which brought it in line with that of Dab + Tram, justified by the fact that the comparator combination has the same mode of delivery.The AC noted the reliance of the results of an economic model on an indirect comparison with Dab + Tram for the comparative clinical effectiveness estimates of Enco + Bini. The company’s model included the outputs of the NMAs, which included small numerical differences in estimates of PFS, OS, AEs and HRQoL in Enco + Bini versus Dab + Tram. The ERG suggested a cost-minimisation approach to the analysis, assuming clinical equivalence, as the company’s NMA did not demonstrate any significant differences in investigator-assessed PFS, OS, AEs or HRQoL between the combination treatments Enco + Bini and Dab + Tram. The clinical experts advised the AC that similar efficacy between the two combination treatments is biologically plausible. The AC was concerned that the absence of evidence does not equate to evidence of absence, and therefore clinical equivalence is not proven by the company’s indirect comparisons. Given that the numerical differences (from the company’s NMA results) included in the company economic model were small, the AC concluded that both the assumptions made in the company base-case analysis of a difference in effectiveness between Enco + Bini and Dab + Tram and the ERG’s cost-minimisation approach were appropriate for decision-making.The AC were presented with the economic analyses results using confidential commercial arrangements for all four drugs in the treatment combinations Enco + Bini and Dab + Tram.The AC recommended Enco + Bini for the treatment of adult patients with unresectable or metastatic melanoma with a BRAF V600 mutation. The final guidance was published by NICE in February 2019 [There is no direct evidence to confirm any differences in clinical effectiveness between Enco + Bini, and Dab + Tram and the available indirect evidence is unreliable. As a result, the AC agreed that a cost-minimisation approach was an appropriate method to compare the treatment options.There is no evidence of the clinical and cost-effectiveness of Enco + Bini versus immunotherapies, such as nivolumab and pembrolizumab. Immunotherapies are recommended for use in all patients, regardless of mutation status; however, there is no consensus as to the optimal approach to the sequencing of BRAF-targeted therapies and immunotherapies. Clinical advice to the AC was that the outcomes for patients would be similar regardless of treatment sequencing. Comparisons between Enco + Bini and immunotherapies were not the focus of this appraisal.Commercial access arrangements are in place for both treatment options, and therefore the incremental cost-effectiveness ratios per QALY gained are confidential; however, the AC was satisfied that Enco + Bini was likely to be a cost-effective use of NHS resources and therefore recommended its use in patients with BRAF V600 mutation-positive unresectable or metastatic melanoma.