Plasma insulin clearance is an important determinant of plasma insulin concentration. In this review, we provide an overview of the factors that regulate insulin removal from plasma and discuss the interrelationships among plasma insulin clearance, excess adiposity, insulin sensitivity, and type 2 diabetes (T2D). We conclude with the perspective that the commonly observed lower insulin clearance rate in people with obesity, compared with lean people, is not a compensatory response to insulin resistance but occurs because insulin sensitivity and insulin clearance are mechanistically, directly linked. Furthermore, insulin clearance decreases postprandially because of the marked increase in insulin delivery to tissues that clear insulin. The commonly observed high postprandial insulin clearance in people with obesity and T2D likely results from the relatively low insulin secretion rate, not an impaired adaptation of tissues that clear insulin. Compared with healthy lean people, people with obesity have increased basal and postprandial plasma insulin concentrations ,2,3. PeoInsulin is produced by pancreatic β cells and secreted into the portal vein, which delivers the newly produced insulin to the liver. The liver extracts approximately half or more of the newly secreted insulin ,11,12; tFor insulin to be taken up by the liver, skeletal muscles, and adipose tissue, it must first pass through the endothelial cell layer of capillaries into the interstitium . The livThe rate and extent of insulin receptor endocytosis is directly related to the insulin concentration; the higher the insulin dose, the faster the internalization process and the greater the number of receptors that are trapped inside cells . Once inThe removal of insulin from plasma in vivo is quantitated in multiple ways that provide unique and complementary insights into the insulin removal process. Plasma insulin clearance rate refers to the amount of plasma that is cleared of insulin per unit of time and is expressed as liters/minute. Insulin extraction or uptake rate refers to the molar amount of insulin that is removed from plasma per unit of time; it represents the product of insulin clearance rate and plasma insulin concentration and is expressed as pmol/minute. Therefore, the insulin extraction or uptake rate can be high even if the plasma insulin clearance rate is low, or vice versa, the extraction or uptake rate can be low even if the clearance rate is high . InsulinHighly sophisticated studies that included arterio-venous blood sampling across various tissues and organs after administering insulin, or after stimulating endogenous insulin secretion, have demonstrated that the fractional extraction of insulin by tissues varies among tissues and is dependent on the insulin delivery rate ,69,70,71The dose-dependent decrease in hepatic and muscle insulin clearance occurs within the postprandial insulin concentration range, but requires at least approximately a doubling or tripling of the basal insulin load. When arterial plasma insulin concentration was increased from approximately 50 pM to 300 pM by infusing insulin, insulin uptake (in pmol/min) by the forearm (muscle) increased, but the fractional extraction of insulin decreased from ~15% to ~5% . Additio15 to 1.0 × 1016 insulin binding sites per liver. If all of the binding sites were on the cell surface and occupied, we estimate the liver could bind at most upwards of 2500 pmol of insulin at once.Collectively, the results from the available studies ,73,74,75The paramount importance of the liver for the removal of endogenously produced insulin in combination with the complex regulation of cellular insulin uptake that is dependent on the insulin delivery rate has important implications for the interpretation of the results from studies that evaluated plasma insulin clearance in people with obesity and T2D. Plasma insulin clearance in people with obesity and T2D has been assessed by using an intravenous glucose tolerance test, an insulin-modified intravenous glucose tolerance test, an insulin suppression test, a hyperinsulinemic or hyperglycemic clamp procedure, an oral glucose tolerance test, and mixed meal tests ,94,95,96Throughout the day, the insulin secretion rate from pancreatic β cells into the portal vein ranges from <200 pmol/min after an overnight fast in lean people to peak values of approximately 800 pmol/min postprandially in people with obesity and insulin resistance ,77. ArteThe intravenous route of insulin delivery during some of the tests , compared with the stimulation of endogenous insulin secretion, also requires consideration. Whole-body clearance of insulin that is administered into a peripheral vein is lower than whole-body clearance of the same amount of insulin appearing in the portal vein after intraportal insulin infusion or glucose-stimulated insulin secretion, because peripherally administered insulin is not subjected to hepatic first pass insulin extraction ,103 Fig. In addiObesity is associated with reduced cell surface insulin receptors in key tissues that are the primary sites of insulin clearance and are also involved in regulating glucose metabolism  ,121,122.Obesity is also associated with reduced hepatic CEACAM 1 expression, and T2D does not alter the relationship between adiposity and CEACAM 1 expression ,135. TheIt is well established that people with obesity have higher basal and postprandial plasma insulin concentrations than lean people ,6,9,139.The effects of obesity, insulin resistance, and T2D on transendothelial insulin transport are unclear because few studies have addressed this topic in people. It has been proposed that insulin transport into the interstitium of muscle tissue is slower in people with obesity and insulin resistance compared with lean people, because the rise in muscle microdialysate insulin after intravenous insulin infusion was slower in obese compared with lean participants ,154. How2 of body surface area) resulted in arterial insulin concentrations that ranged from approximately 300 pmol/L to <1000 pmol/L [Several studies evaluated the relationships among adiposity, insulin sensitivity, and plasma insulin clearance in people with and without T2D by using intravenous insulin infusion protocols (hyperinsulinemic-euglycemic clamp and insulin suppression test) ,92,93,940 pmol/L ,91,92,930 pmol/L ,77. Howe0 pmol/L . However0 pmol/L ,105,106.Although the assessment of insulin clearance during glucose infusion represents the clearance of endogenously produced insulin, and therefore includes an assessment of hepatic first pass insulin extraction, it is not a good substitute for the assessment of postprandial insulin clearance. Intravenously infused glucose does not stimulate the release of incretins, which potentiate the effect of glucose on β cells ,113. InsResults from studies that included lean participants and participants with obesity with different glycemic status, ranging from normal fasting glucose combined with normal glucose tolerance to those with impaired fasting glucose and/or impaired glucose tolerance and T2D, demonstrate plasma insulin clearance correlates with insulin sensitivity ,8,166. TData obtained from a complex mathematical modelling analysis of plasma insulin clearance rates obtained during a series of different glucose ingestion and intravenous glucose and insulin infusion protocols conducted in 2000 lean and obese men and women suggest that there is an inverse relationship between insulin clearance and insulin secretion rate and insulin sensitivity is by far the most important determinant of plasma insulin clearance at any insulin secretion rate . Plasma Non-alcoholic fatty liver disease (NAFLD) is common in people with obesity  and assoPlasma insulin clearance is a highly dynamic, receptor-mediated process and an important determinant of plasma insulin concentration. Basal insulin receptor expression and insulin delivery to tissues that clear insulin are key determinants of insulin clearance. Insulin sensitivity correlates with plasma insulin clearance, presumably because both insulin action in and insulin clearance by tissues require insulin binding to its receptors and insulin receptor expression is downregulated in people with insulin resistance. Moreover, insulin clearance is inversely related to the insulin delivery rate to tissues, so the postprandial increase in insulin secretion itself causes a decrease in insulin clearance and chronic hyperinsulinemia is at least in part responsible for reduced cell surface insulin receptor expression in people with obesity. The higher postprandial insulin clearance in people with T2D, compared to those without, is a consequence of β-cell dysfunction and reduced insulin secretion, which blunts the postprandial insulin delivery rate to tissues that clear insulin. The greater insulin clearance in people with T2D is due to impaired β-cell function, which blunts the postprandial downregulation of insulin clearance caused by insulin receptor internalization after insulin binding.


Accumulating evidence suggests that neurodegenerative diseases are not merely neuronal in nature but comprise multicellular involvement, with astrocytes emerging as key players. The pathomechanisms of several neurodegenerative diseases involve the deposition of misfolded protein aggregates in neurons that have characteristic prion-like behaviours such as template-directed seeding, intercellular propagation, distinct conformational strains and protein-mediated toxicity. The role of astrocytes in dealing with these pathological prion-like protein aggregates and whether their responses either protect from or conspire with the disease process is currently unclear. Here we review the existing literature implicating astrocytes in multiple neurodegenerative proteinopathies with a focus on prion-like behaviour in this context. et al. review the role of astrocytes specifically in prion-like mechanisms of neurodegeneration, across a spectrum of neurological disorders.Astrocytes are increasingly recognised as playing fundamental roles in a range of neurodegenerative diseases. Smethurst  The pathogenic factors underlying the cytotoxic role of aggregated and misfolded proteins are predicted by the prion hypothesis. There are four main behavioural characteristics that make a protein ‘prion-like’: (i) template-directed seeding and amplification of misfolded proteins to form aggregates; (ii) intercellular spreading of aggregates and propagation of the templated seeding reaction to nearby cells; (iii) conformational strains that produce distinct clinical and biochemical disease entities; and (iv) protein-mediated cellular toxicity.Currently, the differential response of neurons and astrocytes to aggregated proteins with prion-like behaviour remains relatively understudied. Specifically, there is conflicting evidence about the presence of aggregated proteins in astrocytes at post-mortem, how these aggregates affect astrocytes and what role they play in neurodegeneration.in vitro and in vivo for the prion protein.Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases characterized by the deposition of misfolded and aggregated prion protein (PrPSc) in the CNS, often causing severe cerebellar ataxia and rapidly progressive dementia. This is pathologically characterized at post-mortem by spongiosis , reactive transformation of astrocytes, microglial activation and neuronal death. These pathologies result in a clinically heterogeneous set of conditions, whereby certain strains of the protein result in different misfolded conformations of aggregated PrPSc in distinct CNS regions.,PrPSc aggregates were first detected in astrocytes in scrapie infected sheep and were noted to form prior to neuronal aggregates and before the development of the cardinal scrapie pathological features .in vitro models that induce neuronal death,Astrocyte PrPSc infectivity has also been replicated in rodent PRNP) can determine disease susceptibility and disease phenotype in patients with prion diseaseIn the human prion diseases, the presence of a polymorphic variant  at codon 129 of the prion gene  protein as extracellular plaques in the neuropil. Amyloid-β is processed via amyloid precursor protein (APP) at the cell membrane where it is cleaved by gamma secretase into various fragments including Aβ40 and Aβ42. It is now well established that aggregated amyloid-β has prion-like behaviour due to its capacity for seeding, ability to spread and to propagate, form distinct strains and exert toxicity via its aggregated oligomeric intermediates both ,As amyloid-β plaques are extracellular in the human Alzheimer’s disease brain, they do not form in astrocytes. However, astrocytes have well known roles in the secretion, uptake and clearance of aggregated neuronal amyloid-β peptides.,,Reactive transformation of astrocytes is one of the earliest detectable features in Alzheimer’s disease. Astrocyte reactivity is accompanied by the release of pro-inflammatory cytokines such as TNF-α, IL-6 and IL1β, which are thought to mediate neurotoxic effects.,,Mechanistically, this increase in astrocyte reactivity may be driven by Aβ42 oligomer disruption of ER calcium homeostasis leading to ER stress and reactive astrocyte transformation.in vivo via alteration in the secretion of the synaptogenic factor TGF-β1.Amyloid-β oligomers have been found to induce reactivity and reduce neuroprotective capacity in primary astrocyte cultures and The neuroprotective capacity of astrocytes against amyloid-β pathology may lie in some partial uptake of the aggregates for clearance during the early phases of amyloid-β aggregation, but once levels of aggregation are high enough, a neurotoxic role seems to supersede this protection and they become instrumental in the degenerative process. However, the role of astrocytes in the processing, seeding and transmission of amyloid-β pathology remains incompletely resolved and requires further investigation.,The term tauopathy refers to a collection of progressive age-related neurodegenerative diseases that are clinically and biochemically heterogeneous, but commonly involve the deposition of misfolded, aggregated and phosphorylated tau inclusions in the cells of the CNS. The tau protein aggregates in the form of neurofibrillary tangles (NFTs), pre-tangle deposits and neuropil threads within neuronal and astrocytic processes. The primary tauopathies include frontotemporal lobar degeneration (FTLD) with tau, corticobasal degeneration (CBD), Pick’s disease (PiD) and progressive supranuclear palsy (PSP), each of which can express various isoform ratios (3R and 4R), conformations and subcellular localization of tau pathology. While tau is predominantly expressed in neuronsMAPT gene mutations that can cause other tauopathies.Alzheimer’s disease-related tau pathology may be distinct from other tauopathies given that tau aggregation in this context is considered a secondary event to the deposition of extracellular amyloid-β plaques. Furthermore, wild-type tau aggregation is present in Alzheimer’s disease-related tau pathology rather than tau aggregation driven by Astrocytes exhibit a range of morphological phenotypes across different tauopathies including tufted astrocytes in PSP, astrocytic plaques in CBD, thorn shaped astrocytes, granular fuzzy astrocytes in ageing-related tau astrogliopathy (ARTAG), ramified astrocytes in PiD and astrocytes with globular inclusions in globular glial tauopathy. One of the first studies to note paired helical filament or phospho-tau S396/S404 positive tau pathology in aged mice demonstrated that astrocytes may be involved in the uptake and spread of tau pathology.,in vitro and in vivo models of tau spread with a significant role in astrocytic  tau uptake.in vitro co-culture models and in vivo models will shed more light on the temporal role of astrocytes in the pathogenesis in the tauopathies.The effects of tau inclusions on the health of astrocytes are currently unknown and represent an interesting research avenue for future studies. As glial cells typically do not secrete or express much tau protein, the deposition of tau pathology in glial cells is an unusual phenomenon that may be explained by the uptake of neuron-derived monomeric and aggregated tau from the extracellular space. Uptake of extracellular tau by astrocytes cells can occur via many mechanismsParkinson’s disease, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) constitute a group of neurodegenerative conditions known as synucleinopathies, pathologically represented by the presence of aggregated α-synuclein protein. α-Synuclein is the main component of a pathological hallmark of synucleinopathies known as Lewy bodies and Lewy-like inclusions, found in the neurons of patients with Parkinson’s disease and DLB, and in the astrocytes and oligodendrocytes of patients with MSA.,et al.α-Synuclein is a predominantly synaptic protein that is secreted from neuronal cells by exocytosis.Another study introducing monomeric, oligomeric and fibrillar forms of synuclein to primary rat astrocyte cultures described changes akin to reactive transformation such as morphological changes, and an increase in intracellular ROS, cytokine release and GFAP expression.Collectively, these data present a mixed picture of astrocytes playing both a protective and a deleterious role in response to α-synuclein aggregation. Indeed, many of these observations can potentially be attributed to differences in the models used for each study including species (both of cell model and inoculum), astrocyte subtype and duration of treatment. The key to elucidating the role of astrocytes in α-synuclein aggregation induced toxicity lies in further investigation using more physiologically relevant cell types such as human iPSC-derived region-specific astrocytes in co-culture with neurons.HTT) that leads to the development of a movement disorder, psychiatric and cognitive symptoms. Translated polyglutamine expansions in the huntingtin protein, if higher than 35 repeats, lead to the aggregation of huntingtin (HTT) protein and the development of Huntington’s disease. A pathological hallmark of Huntington’s disease is the presence of aggregated HTT in the form of neuronal intranuclear inclusions located in the striatum. It is thought that such pathology causes the substantial degeneration of medium spiny neurons, but HTT aggregation has also been identified in astrocytes, and its role in these cells as part of the disease process is still not clear.,,in vitro cellular models , and aberrant electrophysiological responses.Early evidence suggested that glial expression of mutant HTT containing polyQ expansion of 115–150 repeats induces its accumulation in glial nuclei in Huntington’s disease brains, accompanied by a decrease in glutamate transporters and glutamate uptake in glial cultures and mouse Huntington’s disease brains.Drosophila model of Huntington’s disease.Drosophila orthologue of MEGF10 in mammals, and is an engulfment receptor that regulates the clearance of cellular debris. These data indicate that glia may be contributing to the clearance and transmission of aggregates throughout the CNS and that modulating MEGF10 signalling may be a potential therapeutic target for reducing spread. Another study using a mouse model with basal ganglia restricted expression of N-terminal HTT fragments in either astrocytes, neurons or both demonstrated that astrocytes are less vulnerable to HTT aggregates. The study also showed that mutant HTT in neurons affected key components of the glial glutamate-glutamine cycle, at least at the mRNA level.,The prion-like transmission of HTT aggregates from neurons to glia was first demonstrated in a TARDBP gene are also causative of a small percentage of ALS and FTLD cases giving TDP-43 a central role in the pathogenesis of ALS and FTLD.Trans active DNA response binding protein (TARDBP) with a molecular weight of 43 kDa (TDP-43) is a DNA and RNA binding protein. Its abnormal cytoplasmic aggregation is considered the pathological hallmark of ∼97% of cases of ALS and ∼50% of cases of fronto-temporal lobar degeneration (FTLD-TDP), which have now been reclassified as TDP-43 proteinopathies. Mutations in the GFAP,In vitro, human iPSC-derived astrocytes with a TDP-43 M337V mutation have been reported to show cytoplasmic TDP-43 mislocalization and a cell autonomous survival phenotype.TDP-43 is a predominantly nuclear protein that can dynamically shuttle between the nucleus and the cytoplasm. In ALS and FTLD it becomes mislocalized to the cytoplasm where it is cleaved, hyperphosphorylated and aggregated. The presence of aggregated TDP-43 is well documented in neurons and is also a common feature in oligodendrocytes.There is mounting evidence to demonstrate that TDP-43 is also a prion-like protein demonstrating many features of prion-like behaviour,Multiple lines of evidence discussed herein implicate astrocytes as key players in proteinopathies in terms of their roles in developing/uptaking, transmitting and clearing pathological protein aggregates in multiple neurodegenerative diseases . In addiThis work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK 65 (FC010110), the UK Medical Research Council (FC010110) and the Wellcome Trust (FC010110). R.P. holds an MRC Senior Clinical Fellowship (MR/S006591/1) and a Lister Research Prize Fellowship.The authors report no competing interests.


Meniere’s disease is an inner ear disorder characterized by the presence of endolymphatic hydrops in the inner ear and symptomatology of recurrent and debilitating vertigo attacks, tinnitus, aural fullness, and fluctuating sensorineural hearing loss. Although many therapeutic options for MD have been proposed during years, no consensus has been reached by the scientific community. In the last decade, many therapeutic options have been proposed, as intratympanic steroid, intratympanic gentamicin, and intravenous glycerol. Recently, the role of the antisecretory factor in the diet of MD patients have been investigated. Surgery is recommended for intractable MD; some authors proposed new approaches including transcanal endoscopic infracochlear vestibular neurectomy, new marsupiliazation technique in sac surgery, and tenotomy of the stapedius and tensor tympani muscles. Although hearing loss is mainly sensorineural, low-frequency air-bone gap (ABG) can also be found in the absence of middle ear pathology4, mimicking other conditions such as cerebral vascular anomalies including dural arteriovenous fistula5. MD symptoms can severely affect the quality of life7.Meniere disease (MD) is an inner ear disorder characterized by the presence of endolymphatic hydrops (EH) in the inner ear and symptomatology of recurrent and debilitating vertigo attacks, tinnitus, aural fullness, and fluctuating sensorineural hearing loss (SNHL)11. First-line treatment includes dietary restrictions for salt, caffeine, and alcohol associated to drugs both for acute attacks 12, and as prophylactic therapy 13.Although many therapeutic options for MD have been proposed, no consensus has been reached by the scientific community14; however, many studies showed that IT gentamicin may expose the patients to a risk of hearing loss, depending on dosage and intervals between administrations15. Labyrinthectomy or other surgical procedures can be suggested for intractable MD16.When first-line treatment does not offer satisfactory symptom control, intratympanic (IT) administration of gentamicin or corticosteroids can be performed20, scientific research for MD is a growing area in continuous development. This brief review aims to assess the clinical innovations for the therapy of MD.Despite the recent COVID-19 pandemicThe main goal of the pharmacological treatment of MD is to reduce the duration and frequency of vertigo attacks, and to prevent hearing loss, which is usually progressive. There’s lack of high-level evidences for efficacy of MD treatment; for this reason, different drugs and different methods of administration have being tested.21. Pradhan et al22 evaluated the effects of IT dexamethasone in 30 patients with refractory MD comparing the pre- and post-treatment hearing outcome and dizziness score. They found that 23% of patients were free of vertigo at the end of 24 months after IT steroids, but there was no significant improvement in hearing. These results are in accordance with those from Weckel et al23 who retrospectively evaluated 25 patients treated with IT dexamethasone; they reported a satisfactory control of vertigo in 92% of the patients, and this protocol achieved control of vertigo in 70% of patients at 2 years. Harcourt et al24 suggested IT methylprednisolone as an effective treatment for the long-term control of vertigo attacks, without he known inner-ear toxicity associated with gentamicin.IT steroids  treatments have been widely used for MD, especially when first-line treatment does not offer a satisfactory symptoms control; however, there is still lack of evidence that this treatment may have a positive effect in MD25, has been proven as an effective treatment for vertigo control in MD with a potential risk of herring loss; however, discussion about the dosage and the method used is open26. Scarpa et al27 proposed a low-dose IT gentamicin protocol; they treated 48 patients with 0.5 ml of 10 mg of gentamicin with an interval of 2 weeks between injections. They reported a satisfactory control of vertigo attacks after IT gentamicin, and the effect of this protocol on vestibular system was proved by the reduction in vestibulo-ocular reflex (VOR) gain in the affected side. A meta-analysis from Jian et al28 demonstrated that IT gentamicin is superior to IT steroids in reducing the number of vertigo attacks, but both drugs didn’t show hearing improvement. Moreover, Ozturk and Ata proposed an IT mixture of gentamicin and dexamethasone injection for the treatment of intractable MD, even more effective that IT dexamethasone for vertigo control29.Gentamicin, administered at different doses and timing30; in addition, a meta-analysis from Rosenbaum and Winter31 concluded that it is not clear if diuretic lead to a symptomatic improvement of vertigo.Diuretics have also been recommended to control vertigo attacks in MD, due to their effects in reducing the endolymphatic pressure and volume. As for steroids and gentamicin, there are no high-quality data demonstrating the efficacy of oral diuretic therapy32 evaluated the effectiveness on vertigo control of intravenous (IV) glycerol (an osmotic diuretic), 10% glycerol with 0.9% sodium chloride, 0.5 g/kg once a day for 2 consecutive days every fifteen days for six months; the authors reported an improvement of vertigo attacks and a significant reduction of discomfort generated by tinnitus, raising quality of life.A prospective study from Scarpa et al33. It has been hypothesized that AF can act as a modulator of water and ions and interact with the aquaporins. Viola et al34 observed a significant improvement in daily activities in patients treated with specially processed cereals (SPC) compared to those treated with IV glycerol and dexamethasone. The study reported a significant reduction of vertigo spells and a positive effect on tinnitus severity.Recently, some authors suggested a possible action of anti-secretory factor (AF), a protein produced by pituitary gland that plays a role in the innate defense against the inflammatory and secretory components of diarrheal disease35.According to international guidelines, surgical therapy for MD is recommended only for refractory disease, and therefore represents the third (or even the fifth) line of management. Currently, the most popular surgical procedures to control vertigo attacks in MD are also the most aggressive and those that have the most negative impact on auditory function. Besides, there is a lack of evidence suggesting that surgical therapy for MD may provide a significant control of symptomsThe table below shows the main surgical procedures for MD taking into account of hearing preservation, vestibular injury, surgical difficulty and long-term outcomes .36. Xu et al37 suggested that ESS with posterior tympanotomy and local steroid treatment could improve hearing and ensure a satisfactory vertigo control in patients with intractable MD. In Gibson et al opinion38, ESS can provide a vertigo control at least as well as IT gentamicin with a lower incidence of audio-vestibular complications39. Transmastoid labyrinthectomy alone40 or in combination with cochlear implantation41, grommet insertion42, and triple semicircular canal plugging43 have been proposed as an effective surgical option for the treatment of MD.Endolymphatic sac surgery (ESS) can be considered in patients that are refractory to medical management as a nondestructive option, as the risk to damage hearing is low44 evaluated the results of a new marsupialization technique in endolymphatic sac decompression surgery, in which the outer layer of the sac was turned around and placed under the anterior bony border. They concluded that this technique can control progressive hearing loss and improve tinnitus, vertigo, and ear fullness among patients with intractable unilateral MD.New techniques have been recently described. Daneshi et al45 proposed a transcanal endoscopic infracochlear vestibular neurectomy on cadaver as a minimally invasive approach to distal vestibular neurectomy.Moreover, Trakimas et al46.More recently, some authors suggested that tenotomy of the stapedius and tensor tympani muscles (TSTM) may be a safe surgical procedure with significant vertigo control, decreased postoperative symptoms and important hearing preservationIn the diagnosis and the evaluation of therapy efficacy, the audio-vestibular test battery has a primary role, and the diagnostic findings are constantly evolving.47.Di Stadio et al proposed head-shaking nystagmus (HSNy) as a useful test in the early stages of MD to predict a new vertigo attack, with a sensitivity of the ipsilesional HSNy of 100% at T0 and 85.7% at T148 evaluated the VOR performance during the attacks of MD using video head-impulse tests (video-HITs) according to each ictal phase; the authros suggested that during the vertigo attack, HITs are normal during irritative and recovery phases, but abnormal in 53% of the patients during the paretic phase, reflecting characteristic ictal vestibular discharges. Besides, a common finding in MD is a normal horizontal HIT with an asymmetric caloric function, and this dissociation can be used as a diagnostic marker49.Lee SU et al50.Patients with suspected MD can be examined using magnetic resonance imaging (MRI) to evaluate a possible inner ear disease. The MRI findings in patients with MD are conflicting, due to the MRI sequences used and the inclusion criteria of the patients; also, the early stage of MD and the early symptoms appear too subtle for identification using MRI, making the reproducibility of hydrops MRI scan protocols debatableDespite accumulating evidence for the treatment of MD, there is currently no international consensus on a standardized therapeutic protocol for this condition. Intravenous administration of glycerol, low-dose intratympanic gentamicin, and antisecretory factor are promising innovations.The role of surgery in the treatment of MD is still debated; recent techniques as tenotomy of the stapedius and tensor tympani muscles seems to offer interesting insights, but larger randomized studies are needed to draw conclusions.


In this review, we analyze medical and select ethical aspects of the increasing use of next-generation sequencing (NGS) based tests in newborn medicine. In the last five years, there have been several studies exploring the role of rapid exome sequencing (ES) and genome sequencing (GS) in critically ill newborns. While the advantages include a high diagnostic yield with potential changes in interventions, there have been ethical dilemmas surrounding consent, information about adult-onset diseases and resolution of variants of uncertain significance. Another active area of research includes a cohort of studies funded under Newborn Sequencing in Genomic Medicine and Public Health pertaining to the use of ES and GS in newborn screening (NBS). While these techniques may allow for screening for several genetic disorders that do not have a detectable biochemical marker, the high costs and long turnaround times of these tests are barriers in their utilization as public health screening tests. Discordant results between conventional NBS and ES-based NBS, as well as challenges with consent, are other potential pitfalls of this approach. Please see the Bush, Al-Hertani and Bodamer article in this Special Issue for the broader scope and further discussion. With the rapid evolution of molecular genetics, next-generation sequencing (NGS) testing methods such as exome sequencing (ES) and genome sequencing (GS) are being increasingly used for diagnostic evaluation of critically ill infants as well as for newborn screening (NBS) ,2,3. TheIn the past few years, there have been several studies by various groups to explore the use of NGS-based tests such as ES and GS for NBS. While these techniques may enable clinicians to screen for rare diseases that do not have any reliable biochemical markers, there are several moral caveats to be considered. ,2,4.NBS was designed as a universal cost-effective screening test aimed at early detection of treatable hereditary (and a few non-hereditary) conditions to prevent long-term mortality and morbidity . The tesRobert Guthrie devoted several years to developing the first screening test utilizing the bacterial inhibition assay for phenylketonuria . The NBSNorth Carolina Newborn Exome Sequencing for Universal Screening (NC NEXUS) enrolled 106 newborns and children with previously diagnosed metabolic diseases and hearing loss, and ES results confirmed an underlying diagnosis in 88% and 18% of the patients, respectively. The results included pathogenic variants in hereditary cancer syndromes in two children, and 1.8 variants per patient showing a carrier status for recessive conditions ,4.Utilization of ES-based NBS is advantageous as it allows for screening of several monogenic conditions that do not have a consistent analyte that can be measured using MS/MS. For instance, mitochondrial disorders and proximal urea cycle disorders have historically been impossible to screen, despite the availability of therapies and interventions ,17. ES-bWhile shortening the diagnostic journey for infants who are sick can substantially change family’s viewpoints, diagnosis of childhood-onset conditions and carrier status can cause untold and unwarranted anxiety ,3,4. TheWhile the newborn screening programs and the BabySeq project have focused on predicting and preventing future disease in pre-symptomatic infants with genetic disorders, another active area of investigation has focused on the use of rapid genomic sequencing techniques in the evaluation of critically ill infants.Studies have estimated that genetic diseases are present in approximately 16% of neonates in regional ICUs; furthermore, there is high mortality in infants with genetic diseases, which accounts for an estimated 20% of deaths in this age group, and in one center, accounted for an estimated 45% of neonatal intensive care unit (NICU) deaths over a ten-year period ,26. It iSince 2015, several studies have sought to investigate the clinical utility of genomic sequencing in the NICU setting . In thesThe majority of these studies have quantified changes in management as a secondary measure and have shown repeatedly that these NGS results and subsequent genetic diagnoses have a direct impact on the decisions made by clinicians and families. These changes have encompassed both escalation in patient care , as well as limitations in care  ,31,32,34BRCA2 mutation in a male infant whose parents had not consented to receive information regarding adult-onset disease [BRCA2 mutation, the potential impact of this autosomal, dominant adult-onset condition on the health and mortality of the child’s affected parent was considered a threat to the wellbeing of the family unit, which posed a credible downstream threat to the patient’s health as well [While the feasibility of rapid genomic sequencing for the timely diagnosis of suspected monogenic disorders in critically ill newborns has emerged only over the last decade, much accompanying literature over the same time period explores the potential technical and ethical concerns that may limit the widespread adoption of this testing approach. These include the current payer system that typically reimburses only outpatient genomic studies, the challenge of timely resolution of variants of unknown significance (VUS) and the possible impacts of rapid transition to genetic testing on parental anxiety and subsequent parent-infant bonding ,37. One  disease . As a re disease . While s disease ,40. With as well .Another ethical consideration is access to these technologies, which is limited by several factors, including availability of in-house testing and the associated costs of genomic testing in the setting of uncertainty regarding the likelihood of insurance reimbursement for these testing strategies. Several studies have suggested that panel-based testing could prove a reasonable alternative strategy to accomplish rapid diagnosis in the ICU setting, with the proposed benefits of ensuring that a greater number of patients in need of these rapid diagnoses receive them, while also decreasing costs. In one study, a panel of 4503 genes was utilized for rapid evaluation of critically ill neonates; in the study, this panel-based approach yielded a diagnosis in 10/20 (50%) cases and a partial diagnosis in an additional 1/20 (5%) cases, similar to those results reported in the genomic sequencing studies referenced in As sequencing platforms and informatic systems designed to quickly analyze large amounts of genomic data become increasingly agile, and as these testing modalities become increasingly available through commercial means, the likelihood that institutions will transition toward genomic sequencing as a first-line genetic testing strategy continues to increase. As such, our resolve to tackle and mitigate the ethical concerns that accompany these testing modalities must continue to strengthen as well.…whether you like it or not, a complete sequencing of newborns is not far away” [This paper reviewed select ethical aspects regarding the use of ES and GS in NBS and in the diagnostic evaluation of critically ill newborns . The clinical utility and cost-effectiveness of NGS-based NBS are yet to be established. Dr. Francis Collins, NIH Director, has remarked: “ar away” . Genomicar away” ,46,47.


Precision oncology can be defined as molecular profiling of tumors to identify targetable alterations. Emerging research reports the high mortality rates associated with type II endometrial cancer in black women and with prostate cancer in men of African ancestry. The lack of adequate genetic reference information from the African genome is one of the major obstacles in exploring the benefits of precision oncology in the African context. Whilst external factors such as the geography, environment, health-care access and socio-economic status may contribute greatly towards the disparities observed in type II endometrial and prostate cancers in black populations compared to Caucasians, the contribution of African ancestry to the contribution of genetics to the etiology of these cancers cannot be ignored. Non-coding RNAs (ncRNAs) continue to emerge as important regulators of gene expression and the key molecular pathways involved in tumorigenesis. Particular attention is focused on activated/repressed genes and associated pathways, while the redundant pathways (pathways that have the same outcome or activate the same downstream effectors) are often ignored. However, comprehensive evidence to understand the relationship between type II endometrial cancer, prostate cancer and African ancestry remains poorly understood. The sub-Saharan African (SSA) region has both the highest incidence and mortality of both type II endometrial and prostate cancers. Understanding how the entire transcriptomic landscape of these two reproductive cancers is regulated by ncRNAs in an African cohort may help elucidate the relationship between race and pathological disparities of these two diseases. This review focuses on global disparities in medicine, PCa and ECa. The role of precision oncology in PCa and ECa in the African population will also be discussed. Sir William Osler 1849–1919) famously stated that no two persons can react alike to disease 849–1919 ,3. The cPrecision medicine relies on how an individual’s unique molecular profile influences the individual’s susceptibility to disease and response to medical treatment . Over thThe World Bank defines low middle income countries as those where the Gross National income (GNI) is between $1036 and $4045 per capita, while the High-income countries are defined as those with a GNI between $4046 and $12,535. These labels are those set by the World Bank in 2021 and are regularly re-assessed based on the changing economy of the world . ProstatAn analysis of the incidence rates of PCa in the GLOBOCAN database  shows thGLOBOCAN includes endometrial cancers under the umbrella term Corpus Uterine cancers . EndometProstate cancer is often only diagnosed at a very late stage because the initial stages of the disease have no symptoms . If sympOnce these initial studies have indicated the presence of PCa, further diagnostic tests can be carried out and these include transrectal ultrasound, magnetic resonance imaging (MRI) or prostate biopsy. These techniques require specialised equipment and trained practitioners and may be limited in LMICs . The aggEnvironmental factors such as diet have been implicated in contributing to PCa incidence. Men who emigrate from a country with a low incidence of PCa tend to develop PCa at the same rate as men in their adopted country. This implicates the change in environment between the native low incidence country to that of the high incidence adopted country as a risk factor . One of Therapeutic clinical trials for men with PCa have considerably increased over the past 16 years . HoweverThe United Nations (UN) has developed the Human Development Index (HDI) as a statistic to measure a country’s level of social and economic development. The HDI is made up of various measurements, The mean years of education, life expectancy and gross national income per capita . A studyKLK2/3 (19q13.33), NUDT10/11 (Xp11.22) [HNF1B/TCF2 (17q12) [JAZF1, and MSMB [In precision oncology, the best interpretation of genomic results is achieved through multidisciplinary input. This also reduces bias and uncertainty in the clinical data . Many geXp11.22) , 11q13.2 (17q12) , JAZF1, and MSMB . Table 1AR), telomerase-related genes  [(UGT1A8) and cytochrome P45021A2 (CYP21A2), metalloproteinase genes and various non-coding RNAS (ncRNAs), including micro-RNAs (miRNAs) and long non coding RNAs (lncRNAs) [FOXA1) mutations [Previous studies involving varied populations, but still mainly European men, have identified genes that are associated with PCa include those involved in DNA damage and repair, carcinogen metabolism, inflammation and steroid hormone metabolism ,63,64,65RT, TET) , genes ilncRNAs) . By studutations . FOXA1 iutations .A similar study comparing mutation profiles in men of African and European ancestry with PCa was conducted. This study focused on genes involved in immune-oncogenic pathways. A race specific gene expression profile was identified with 38 differentially expressed genes specific to each race group. These genes were involved in immune-oncogenomic pathways such as cytokine signaling, interferon (IFN) signaling (IFNγ and IFNα responses), apoptosis, nuclear factor NF-kB (NF-kB) signaling in the tumor necrosis factor alpha (TNFα) pathway, epithelial–mesenchymal transition (EMT) pathways, and signaling by ILs including IL4 and IL13. This implies that immune related pathway alteration is more prevalent in men of African ancestry with PCa .Testosterone metabolism plays an important role in PCa and the cytochrome P450 enzymes involved in this metabolic pathway show high levels of allelic variations depending on ethnicity and ancestry ,71. The The symptoms of endometrial cancer include post-menopausal bleeding, bleeding between periods, pelvic pain and abnormal discharge. It is advised that women with these symptoms should be screened, but there is no evidence that asymptomatic women should be screened . High riEven in higher income countries, such as the U.S.A., patient mortality and positive treatment outcome rely on the early diagnosis of endometrial cancer. For instance, a comparison between the mortality rates in rural and urban endometrial cancer patients in Utah showed that rural women had a higher mortality rate as a result of a later diagnosis of the disease. This is most likely due to screening facilities not being readily available in rural areas, requiring patients to take more time and effort, travelling a greater distance .In African American communities, the best predictors of endometrial cancer related death  were early stage diagnosis. This was followed by family income and the financial well-being of the family and body mass index (BMI). However, further analysis revealed that the only stage of the cancer and BMI are accurate predictors of patient survival . BMI is Histologically, ECa is classified into either type I, estrogen dependent with a better outcome or type II, estrogen-independent with a poor prognosis. However, recently, histopathological and molecular reports have indicated a rather complex ECa risk stratification approach . ResultsAlthough recent advances have identified a number of molecular targets, which are currently being explored for effective treatment of type II ECa, there is still a lack of novel biomarkers and therapeutic targets. P53 mutations have been reported in 57.7–92% of type II ECa .KRAS), human epidermal growth factor receptor 2 (HER2), epithelial growth factor receptor (EGFR), phosphatidylinositol 3-kinase catalytic subunit (PI3KCA) and fibroblast growth factor receptor 2 (FGFR2). Additionally, the expression of tumor suppressors, such as PTEN, p53, p21 and cyclin-dependent kinase inhibitor 2A (CDKN2A), is also altered in type II ECa. Other genes whose expression is altered in type II ECa include genes involved in apoptosis, genes involved in DNA mismatch repair and genes coding for hormone receptors ) [Studies have identified multiple genes whose expression changes in type II ECa. Oncogenes whose expression changes include GTPase kras (and PR)) ,89.The change in the expression of some genes is so marked that they can be used as markers for ECa; these include the proliferation marker Ki-67 and angiogenesis growth factors (VEGF-A) . More ECMicroRNAs (miRNAs) act as regulators of gene expression by binding to specific mRNAs and inhibiting or modifying their translation. They are able to target specific mRNAs by binding to complementary regions in the 3’ untranslated region (UTR) of the target mRNAs  Figure . AberranMCL1, miR-513c/STAT1, miR-96/FOXO3A, miR-145/ITPR2, and miR-34a/PPP2R2A. The results of the study also suggested that the changes in these miRNAs activate EGFR–PI3K–AKT signaling pathways while knockdown of these miRNAs results in decreased proliferation, aggression and sensitivity to docetaxel-induced cytotoxicity [The PCa differences in the miRNA profiles of men of European ancestry and men of African ancestry have been identified that are related to the occurrence, prognosis and progression of PCa . Five mitoxicity .A study examining the differences in miRNA profiles between healthy women and women with type II ECa, identified 280 miRNAs whose expression was different in the two groups. Women of African ancestry with ECa had increased expression of miR-1269b and decreased expression of miR-1269a, miR-891a and miR-892a compared to women of European ancestry . The miRDD3), also known as prostate cancer antigen 3 (PCA3), is an lncRNA that was shown to be significantly overexpressed in PCa [Long non-coding RNAs (lncRNAs), which are transcripts that are >200 nucleotides long and have no protein-coding potential, have emerged as important targets in tumorigenesis and tumor progression studies. Aberrations in the transcription profiles of various lncRNAs have been shown to be the driving force behind several cancer phenotypes . They dod in PCa . It has d in PCa .In endometrial cancer, the lncRNA H19 has been shown to be significantly overexpressed in 60% of EC . Its expThe use of lncRNA as biomarkers and therapeutic targets for PCa and type II ECa in individuals with African ancestry has great potential in precision oncology ,93.Most clinical trial studies have been done in patients from high-income countries (HICs). Furthermore, as previously discussed, emerging evidence indicates that, in addition to the environmental factors, genetic ancestry and geographical factors also contribute to the diverse molecular landscape of disease . In addiThis suggests that there may be significant genetic differences in tumor types found in HICs and LMICs ,119,120.Even though studies from HICs reveal major differences in toxicities and the response to treatment across various ethnic groups, genomic data from LMICs are still scarce. Differences in polymorphisms across different ethnic groups also play a role in precision oncology . For exaDespite efforts made by the National Institutes of Health (NIH) Revitalization Act in 1993, instructing the inclusion of underrepresented/minority populations and women in clinical trials, persistent exclusion or minimal inclusion of such minority groups/populations continue to arise .The reluctance of individuals from specific population groups to participate in research and clinical trials may partially be attributed to historical unethical practices and fear of exploitation. Awareness campaigns, protection, privacy and transparency policies may also need to be reevaluated. Furthermore, studies illustrate that research in health disparities is inadequately funded ,128,129.Historically, African ancestry linked populations have been underrepresented in clinical trials. Adequate representation of Africans could result in new treatments that would benefit their overall quality of health and prolong their lifespan ,45,46,47Concerted efforts involving clinical, basic and translational research have made significant progress in defeating cancer. However, cancer still remains a public health problem both in developed and LMICs . FurtherThe genomic and molecular investigation of a variety of tumors has initiated the development of drugs targeting the discovered biomarkers. These molecular changes can either be driving the carcinogenic process (driver mutations) or be mere bystanders/passenger mutations ,140. DifPrecision oncology not only focuses on the cancer genome, but also on the transcriptome and proteome of the tumor. This improves the identification of novel biomarkers and therapeutic targets. However, if systemic bias is not properly addressed, such as the inclusion of the minority underrepresented populations, precision oncology may actually exacerbate the disparities that already exist in health care systems . ThroughThe genetic differences between African and non-African populations include copy number variation, haplotype, and nucleotide diversity in mitochondrial and nuclear genomes ,146,147.There is a fundamental need for the inclusion of LMICs, such as those located in the SSA region, in global cancer genomic studies. Apart from benefiting cancer patients in this region, the SSA populations hold enriched genetic diversity which would be instrumental in the continued fight against cancer on a global scale. Even though PCa and type II ECa are linked to African ancestry, much is still unknown. This is why decoding of the genomes of members of this population is necessary to embrace precision oncology. The advantages and implementation of precision oncology are summarized in The burden of PCa on men of African ancestry and ECa type II on women of African ancestry, the great genetic diversity in SSA, the inadequate efficiency of diagnosis-to-cure using conventional treatments for advanced PCa and type II ECa with their known side effects as well as the high costs of some of the more effective treatment strategies available, make the search for affordable, precise medicine an imperative for the African continent. In the pursuit of medical care with a strict adherence to the ethical principle of first do no harm, the clinicians and scientists’ oncology experts are compelled to continue seeking alternatives for the cure and the care of PCa and ECa type II patients in the African region, while being cognitive of the potential associated high costs. At this current point in time, the ability of precision oncology to ease the burden of PCa and ECa type II in the African population may encounter implementation challenges. However, the benefits of this approach will benefit patients of African ancestry in many ways that will justify the time and money spent to develop and implement precision oncology. This calls for the urgent implementation of mechanisms to include populations of African ancestry in international efforts involving genomics to combat heterogeneous diseases such as cancer.


Breast cancer is the most commonly diagnosed cancer in women worldwide. Approximately one-tenth of all patients with advanced breast cancer develop brain metastases resulting in an overall survival rate of fewer than 2 years. The challenges lie in developing new approaches to treat, monitor, and prevent breast cancer brain metastasis (BCBM). This review will provide an overview of BCBM from the integrated perspective of clinicians, researchers, and patient advocates. We will summarize the current management of BCBM, including diagnosis, treatment, and monitoring. We will highlight ongoing translational research for BCBM, including clinical trials and improved detection methods that can become the mainstay for BCBM treatment if they demonstrate efficacy. We will discuss preclinical BCBM research that focuses on the intrinsic properties of breast cancer cells and the influence of the brain microenvironment. Finally, we will spotlight emerging studies and future research needs to improve survival outcomes and preserve the quality of life for patients with BCBM. Central nervous system (CNS) metastases are a devastating diagnosis for patients living with breast cancer. People living with breast cancer and CNS metastasis represent an understudied cohort of patients with unique challenges to manage their disease. CNS metastasis describes any metastases within the brain or the intramedullary spinal cord. This review will highlight the biology, current treatment strategies, and ongoing clinical trials for breast cancer that has metastasized specifically to the brain (BCBM). We will discuss unmet needs that leave patients living with BCBM feeling overlooked.1. Breast cancer is classified using pathological markers, TNM staging , and gene expression patterns1. Breast cancer is broadly classified by origin, either in the breast duct, in the case of intraductal carcinoma (IDC), or the breast lobule for intralobular carcinoma (ILC). Patients with ILC are reported to have a higher likelihood of bone, gastrointestinal and ovarian metastasis and less likely to have CNS, regional lymph nodes or lung metastasis as their first site of metastatic recurrence compared to patients with IDC.Breast cancer is the most common neoplasm among women and causes 500,000 deaths annually worldwide, with ~1.3 million new cases diagnosed each year3 which closely overlap with pathologically defined subtypes. Pathologists use immunohistochemical (IHC) staining to determine the presence or absence of two hormone receptors (HR), the progesterone receptor (PR) and the estrogen receptor (ER), as well as the human epidermal growth factor receptor 2 (HER2)4. IHC-defined tumor subtypes have been associated with a difference in a patients’ median survival at the time of a diagnosis of brain metastasis. Patients with HR+/HER2+, HR−/HER+, HR+/HER2−, and HR−/HER2− have a median survival of 21–27, 18–25, 10–14, and 6–9 months, respectively5. Molecular markers, like BRCA1 or BRCA2 (breast cancer gene 1 or 2) germline mutations, are indicators of possible basal-like breast cancer development and are used to determine potential risk and guide treatment6. Recent large-scale sequencing efforts have led to the identification of the genes with the highest mutation rates in breast cancer, including TP53, PIK3CA, AKT1, PTEN, ERBB2, ATM, CDH1, APC, KRAS, NRAS7. Ongoing research will determine whether these mutations are “actionable” in order to lay the foundation for personalized medicine approaches. The most common organ sites for breast cancer metastases are bone, brain, liver, and lungs9. Breast cancer is the second leading cause of all brain metastasis, and 10–16% of patients with advanced breast cancer have or develop brain metastasis10. National trials are underway to determine if early detection of brain metastasis would improve survival and quality of life, because current NCCN guidelines do not recommend brain imaging unless neurocognitive symptoms develop12. Patients are often not educated about the symptoms suggestive of cancer spread to the CNS. Failure to identify CNS metastasis early likely results in more invasive and toxic interventions such as whole-brain radiation therapy (WBRT) or surgical resection. There is increased support by the patient advocate and medical communities to include brain imaging at the time of a MBC diagnosis, especially in patients with an increased risk of developing BCBM. Koniali et al. provide an extensive review of the risk factors for BCBM13. The main risk factors include age (<49 years old), higher-grade cancer, prior visceral metastases, HER2-positive or triple-negative status, and mutations in the BRCA1 gene.In addition to characterization by origin, breast cancer has been molecularly characterized, initially by five main subtypes 14. Several contributing factors include increased detection due to improved and more widely available radiological techniques and targeted therapies to treat systemic disease, which prolong survival15. The extended survival time has led to a 25–40% increased incidence in brain metastasis in patients with HER2-positive breast cancer and as high as 46% among patients with advanced TNBC17.The incidence of BCBM is rising18. LMD remains understudied because it occurs at a lower rate, is difficult to diagnose20, and is associated with a median survival of 15 weeks20. Le Rhun et al. compared 50 patients with LMD to 50 patients with breast cancer and no CNS metastases including LMD. The cohorts were matched based on their age at time of diagnosis, the year of diagnosis, and the type of chemotherapy that they received. Factors associated with risk for LMD included: lobular histology, HR-negative status, and metastasis at time of breast cancer diagnosis21. In a separate study, patients with TNBC as well as patients with higher grade cancer developed LMD within a shorter time frame as compared to patients’ with receptor positive tumors and/or lower grade tumors22. There are no standards for neurological examinations, neuro-imaging assessment, or a specific CSF cytological to diagnose LMD. A Response Assessment in Neuro-Oncology (RANO) working group has been established to develop these criteria. Finding the means to overcome CNS metastasis is, undoubtedly, an unmet clinical need for which more research is required.BCBM most commonly occurs in the brain’s parenchyma (neurons and glia). The spread of cells to the pia mater, arachnoid mater, subarachnoid spaces, and the cerebral spinal fluid (CSF) is known as leptomeningeal metastasis or disease (LMD)23. Transgenic or knockout mice that develop spontaneous mammary carcinomas that metastasize have also been developed25. An alternative metastasis model was created using variants of human breast cancer cell lines serially injected via the heart or tail of mice and isolated from the bone, lung, or brain26. When tested in experimental metastasis models, by intracardiac injection, these variants showed preferential homing to the organ from which they were harvested27. BCBM experimental models can be established by injecting cells into the mouse brain or carotid artery29, and used to test the ability of therapies to treat CNS lesions effectively. The downside of such models is they circumvent the development of a primary tumor and bypass the initiating steps of metastasis28.Mouse models for breast cancer metastasis rely on the injection of human MBC cell lines or patient-derived organoids into immune-compromised mice. Several mouse cell lines derived from spontaneous mouse tumors are transplantable in a syngeneic murine background with a competent immune system30. The group also discovered that cells in the CSF express complement component 3, promoting disruption of the brain-CSF barrier and predicting leptomeningeal relapse31. Kuruppu et al. established a model in which mice develop neurological symptoms that bear clinical resemblance to LMD. The model can be used to evaluate potential treatment strategies32. Preclinical models that replicate BCBM and LMD are improving, but the lack of spontaneous breast to brain models has stagnated research.Clinically relevant models of LMD were established by Boire et al., who performed three serial rounds of the direct injection of human or mouse cancer cells into the cisterna magna and then collected the primed cells. The primed cells were intracardially injected into a separate cohort of animals, and the disseminated cells consistently formed LMD instead of CNS disease33, a symbiotic relationship of cancer cells with resident cells, a specific gene expression profile, or even the immune microenvironment of the organ35. Whether breast cancer cells have inherent organotropism initially in the primary tumor or whether they adapt to the metastatic site continues to be debated in the field36.Metastasis studies have focused on the concept of “organotropism”, or the ability for a cancer cell to preferentially home to, and survive in, a specific organ. Organotropism could occur due to circulation patterns, a pre-metastatic niche37. The metastases that formed were isolated, dissociated, and underwent multiple in vivo intracardiac injections to select for cells primed to colonize either the lung, bone, or brain37. These CTC-derived brain metastatic cells had high expression of semaphorin (SEM4AD), which increased transmigration in a simulated in vitro blood-brain barrier (BBB) assay and an in vivo mouse model37.In a recent study, circulating tumor cells (CTCs) acquired from four patients with breast cancer were injected into the left cardiac ventricle of NSG mice38. The metastatic brain cells had increased COX2 and EGFR ligand (heparin-binding EGF) expression that promoted BBB permeability via prostaglandin production38. Also, the breast cancer cell surface expressed a brain-specific sialyltransferase (ST6GALNAC5)38. The next step is to combine the results of these studies to develop an assay that can be used to assess the risk of BCBM development.In separate studies, to elucidate mechanisms of extravasation in the brain, nude mice were subjected to serial intracarotid injections of MDA-MB-231 and CN34 cell lines to establish cell lines primed for brain metastasis39. Resident astrocytes and microglial cells express cytokines that promote breast cancer cell proliferation40. Conditioned media from rat neonatal and adult astrocytes enhanced cancer cell invasion in vitro due to secreted matrix metalloprotease-2 (MMP-2) and MMP-9, while inhibiting MMPs in the media decreased metastatic growth of breast cancer cells following intracardiac injection41. This suggests that secreted factors from astrocytes contribute to colonization within the CNS42.Using intravital imaging of intracranially injected MDA-MB-231 cells, Simon et al. observed microglia directly interacting with breast cancer cells, altering microglial morphology and disrupting normal brain electrophysiology43. Cancer cells that adapt to the brain microenvironment by mimicking CNS cells have an increased chance of survival. For example, Neman et al. showed that some breast cancer cells express proteins typically expressed in neuronal cells and can even catabolize GABA into succinate to form NADH44. Furthermore, expression of reelin in HER2-positive cancer cell lines co-cultured with astrocytes had increased proliferation which was reversed with the knockdown of reelin and HER245. In both an intracardiac and intracranial injection models, TNBC and HER2-positive breast cancer cells activated astrocytes by expressing truncated glioma-associated oncogene homolog one, which enhanced brain colonization and increased the expression of genes associated with stemness 46.Astrocyte-derived exosomes can promote chemokine production in breast cancer cells leading to enhanced proliferation and reduced apoptosis47. Another study exploring the interaction of BCBM cells and macrophages demonstrated that cathepsin S is a regulator of BCBM. Inhibiting cathepsin S in both cancer cells as well as macrophages significantly reduced BCBM48. An analysis of metastasis-associated macrophages in the brain parenchyma of mice revealed upregulated cytokine and Lymphotoxin β production that promoted M2 polarization of macrophage cells49. Thus, astrocyte, microglia, and macrophage interactions with breast cancer cells promote metastasis through altered neuroinflammatory responses in the brain50. Basic research has led to the identification of potential targets that could be used to develop therapies or to predict BCBM but to be tested clinically, patients with BCBM need to be included in clinical trials.Microglia/macrophages can directly interact with breast cancer cells that have metastasized to the CNS. For example, Andreou et al. intracerebrally injected 4T-1 cells in BALB/c mice and identified subsets of activated pro-inflammatory and anti-inflammatory microglia/macrophages. The group then selectively depleted the anti-inflammatory microglia/macrophage population using mannosylated clodronate liposomes, reducing brain lesions51. In 2016, ASCO and Friends of Cancer Research (Friends) established a Brain Metastasis Working group to change the exclusionary nature of the current eligibility criteria. The group lobbied for the inclusion of patients with BCBM in trials suggesting that they could be stratified into three cohorts of patients during clinical trial design: (1) those with treated/stable BCBMs, (2) those who have active BCBMs, and (3) those who have LMD52. A fourth cohort consisting of patients that have not received prior treatment but have stable BCBM should be considered. While the ASCO-Friends guidance is welcomed by the patient advocate community, the majority of clinical trials restrict eligibility to those patients with stable BCBM whereas the majority of patients with BCBM have active disease and who are in dire need for a clinical trial. Retrospective and prospective exploratory analysis have been conducted within larger cohorts of patients enrolled in a clinical trial to identify the incidence rate, time to development, and overall survival time following a diagnosis of brain metastasis.Patients with BCBM are often excluded from clinical trials because BCBM is linked with an increased mortality rate. Out of 1474 clinical trials for patients with breast cancer conducted from 1992 to 2016 in a review by Costa et al., only 29% included patients with CNS disease, and only 1% (16 studies) were designed to consider BCBM specificallySeveral treatment approaches are available to patients with BCBM, including local and systemic therapies. The majority of patients diagnosed with BCBM will have one or more local treatments but will likely continue their systemic therapy or transition to a different systemic approach.53. Stereotactic radiosurgery (SRS) is a favorable alternative because of reduced cognitive impairments. Recent Phase III results presented at ASTRO 2020 demonstrated that SRS led to less cognitive decline than conventional WBRT even in patients with multiple lesions (more than 4) without compromising disease control. This serves as the foundation of an ongoing clinical trial comparing SRS with hippocampal-avoidant WBRT plus memantine for 5–15 BM (NCT03550391)54. The study includes patients with active brain metastases but excludes any patients with LMD, >15 BM on a volumetric T1 contrast MRI within the past 14-days or >10 metastasis on non-volumetric MRI. Emerging data suggest that SRS remains a reasonable alternative even for patients with a large number of BCBMs, with one series reporting utilization of SRS in a patient with as many as 34 brain metastases55. A clinical trial of patients with BCBM from non-small cell lung carcinoma, melanoma, and renal cell carcinoma demonstrated a benefit in overall survival from concurrent SRS with immune checkpoint inhibitors, suggesting more studies regarding the timing of SRS in BCBM could provide insight56. Surgery is reserved for patients who present with a limited number of large or symptomatic brain lesion(s). Surgery is typically followed by adjuvant radiation therapy (RT), either in the form of SRS or WBRT57. Following a randomized, controlled, phase III trial (n = 194 patients), post-operative SRS has become the standard of care due to reduced cognitive decline but similar survival benefit when compared to WBRT58. Another treatment option for metastatic brain lesions is laser interstitial thermal therapy (LITT), which remains highly experimental though the technology was established in 1990. It was not until the late 2000s that FDA approved two ablation systems used to treat primary or recurrent tumors and radiation-induced necrosis, which are only available at large medical institutions. LITT is used on deep-seated tumors but its use is limited by the high cost of the procedure60.Localized treatments for BCBM include surgery, WBRT, and radiosurgery. WBRT is preferred when there are many metastatic lesions but does not come without risk. WBRT can cause neurocognitive complications  and does not improve overall survival unless combined with surgery or chemotherapy61. Drugs like memantine, an Alzheimer’s prescription drug, have been utilized to block vascular damage and reduce side effects like dementia that result from WBRT treatment of the hippocampal region62. To assess the neurological impairments that result from radiation treatment, specific cognitive examinations that establish a baseline and evaluate changes should be developed63.Current clinical studies are focused on improving the effectiveness of RT and assessing specific neurological impairments caused by RT. Radiosensitizers like motexafin gadolinium (produces reactive oxygen species), efaproxiral (induces low oxygen by binding to hemoglobin), and RRx-001, which dilates blood vessels and improves oxygenation to the tumor site, have demonstrated efficacy in the prevention of neurocognitive impairment64. Some clinicians are concerned that PCI may be associated with too many adverse effects, including a risk to the patient’s quality of life65. Thus, the application of PCI remains controversial in the field, and clinical trials would be warranted. A partial list of active clinical trials that include a RT component are highlighted in Table Since patients with HER2-positive disease and TNBC have a higher rate of BCBM, prophylactic cranial irradiation (PCI), which has historically been utilized for patients with small-cell lung cancer and brain metastasis, is now being considered66. Doxorubicin, cyclophosphamide, fluorouracil, paclitaxel, docetaxel, and vinorelbine may also be used but have poor blood-brain barrier penetrance11.The systemic treatments often used to manage BCBM include corticosteroids to reduce cerebral edema and standard chemotherapy agents such as capecitabine, carboplatin, gemcitabine, and methotrexateSystemic treatments for patients with BCBM beyond chemotherapy now include a small but quickly growing arsenal of targeted therapies. For example, small molecule inhibitors are being used to treat patients with HER2-positive cancer, and endocrine therapy combined with the cyclin-dependent kinase (CDK)4/6 inhibitor, abemaciclib, for patients with hormone receptor-positive disease.67. Likewise, in the CLEOPATRA trial, an exploratory analysis demonstrated no difference in incidence, but the time to develop CNS metastases was prolonged from 11.9 to 15 months when pertuzumab was added to a trastuzumab and docetaxel treatment regimen68. The NALA trial compared the progression-free survival (PFS) of 101 patients with stable BCBM treated with either neratinib (N = 51) or lapatinib (N = 50) in combination with capecitabine. Patients with BCBM treated with neratinib had a median PFS of 7.8 months compared to 5.5 months for patients treated with lapatinib69. Results from the HER2CLIMB trial show that the median PFS in patients with active BCBM at baseline that received tucatinib, trastuzumab, and capecitabine was 7.6 months compared to 5.4 months in patients who did not receive tucatinib70. In a subgroup analysis of the DESTINY-Breast01 trial, a phase 1 dose-finding study for trastuzumab deruxtecan (T-DXd), 24 of the 184 patients enrolled had stable CNS metastasis. The objective response rate was 58.3%, and median PFS was 18.1 months for these patients71. T-DM1, T-DXd, neratinib, and tucatinib are FDA-approved treatments for patients with HER2-positive MBC that has progressed on prior HER2-targeted therapy(ies).A retrospective analysis of EMILIA trial data showed that the rate of CNS progression was similar , but the median overall survival was significantly improved in patients with asymptomatic CNS metastasis that were treated with trastuzumab emtansine (T-DM1) compared to lapatinib plus capecitabineAt the time of publication, globally, 230 ongoing clinical trials include patients with BCBM of which 36 are open to patients with LMD. The majority of the studies include targeted therapy such as antibody drug conjugates (ADCs), immunotherapies, novel chemotherapeutics, and small molecule inhibitors  cells that have been optimized in a xenograft mouse model of BCBM. The HER2-CAR T cells reduced T-cell exhaustion in vivo and intracranial delivery demonstrated antitumor efficacy74. This research has led to an ongoing clinical trial (NCT03696030) for patients with active brain or leptomeningeal metastases. Other immunotherapies include vaccines that introduce neoantigens that are specific to glioma. When viable neoantigens are identified for BCBM, the same strategy could be employed75.There is a broadening interest in using immune checkpoint inhibitors as therapeutics for BCBM after preliminary studies showed efficacy in other solid tumor types76. The capillaries that make up the BBB are different from the endothelium in other organs. The BBB is composed of endothelial cells with tight junctions, no fenestrations or pinocytic vesicles, and are encased in a basal membrane and extracellular matrix barrier77. Apart from size, polarity (nonpolar preference) and lipophilicity contribute to the restrictions for passive diffusion77. We refer the readers to a review by Deeken and Loscher that discusses ways to overcome the BBB using transporter inhibition, nanoparticles, immunoliposomes, peptide vectors, or carrier-mediated active transport mechanisms77. We also note a retrospective review of animal and human studies of HER2-positive BCBMs by Kabraji et al. that revealed a drug’s ability to cross the BBB did not necessarily correlate with efficacy78, suggesting additional factors contribute to the inadequate response.One main obstacle for identifying efficacious BCBM therapies is the BBB. BCBM development occurs when cancer cells detach from the primary tumor, invade, cross the endothelial barrier, survive in the bloodstream, extravasate, and grow at the secondary site79. For example, a case study reported on a patient with HER2-positive breast cancer and multiple brain lesions treated with intrathecally-delivered trastuzumab, resulting in stabilizing brain and epidural metastases80. A phase 1 clinical trial (NCT01325207) was initiated to test intrathecal delivery of trastuzumab and identify a maximum tolerated dose for patients with LMD and stable systemic disease in HER2+ breast cancer81. Another method of direct CNS delivery is convection-enhanced delivery (CED) via a pressure gradient at the tip of an infusion catheter. Still, this method has not been largely successful due to the dependence on volume and rate of gradient infusion, resulting in an uneven distribution of drugs or potential drug efflux from the injection site and toxicity from treatment82. One final strategy for increasing CNS drug delivery is ultrasound-induced BBB opening that has shown some preclinical efficacy as well as efficacy for CNS diseases such as gliomas82. Intrathecal delivery is largely limited to patients with HER2+ disease as other agents  can cause debilitating toxic side effects with limited benefit and are unsustainable for indefinite use.The direct injection of therapeutics into the spinal canal or subarachnoid space has improved drug efficacy in some instances11. A recent study using AMT-PET imaging successfully differentiated primary brain tumors and metastatic brain tumors with greater than 90% accuracy launching a clinical study for patients with BCBM to improve diagnoses by enhancing the ability to differentiate abnormal and normal tissue (NCT01302821)83. Artificial intelligence (AI) will likely play a future role in assessing treatment response of brain tumors; Machine learning methods carefully trained on standard MRI could be more reliable and precise than established methods84.MRI is most commonly used to monitor disease progression and side effects from treatment, detect recurrence, and identify new metastases post-treatment. Still, current imaging techniques lack the power to differentiate pseudo-progression from actual progression. Improving imaging techniques to diagnose and monitor BCBM earlier could enhance the quality of life for patients85. Riebensahm et al. showed a significant association of decreased overall survival when CTCs were detectable86. Cell-free DNA (cfDNA) has been used to assess genomic alterations. To identify genetic mutations, cfDNA was isolated and sequenced in blood samples from 13 patients with BCBM and 36 patients without BCBM87. There was a high correlation of mutations in APC, BRCA1, and CDKN2A associated with BCBM, which provided supportive evidence for cfDNA biomarkers88. In a study of 194 patients with MBC cfDNA and CTCs were compared for their ability to predict PFS, OS, and response to treatment. cfDNA was simpler to isolate, more informative, and less expensive than isolating and quantifying the number of CTCs89. Proteins in the serum or CSF of patients have also been considered as predictive biomarkers of metastasis. For example, Dao et al. identified increased (>0.1 ng/mL) levels of the angiopoietin-like fibrinogen-like domain (cANGPTL4) in the serum of patients with breast cancer, which was associated with increased risk for BCBM90.To identify patients most at risk to develop BCBM, researchers are advancing the capabilities of liquid biopsies since actual BCBM biopsies are an impossibility in most cases91.Due to a lack of guidelines for the treatment of CNS metastasis, once a diagnosis is confirmed, treatment is at the discretion of a patient’s oncology team. Most cancer patients are treated locally in community hospitals. They are not likely to have access to a breast oncologist specializing in CNS metastasis or a multidisciplinary team who form a consensus on treatment decisions. Even with access to a multidisciplinary team, which often includes a medical oncologist, neuro-radiation oncologist, neuro-oncologist, and neurosurgeon, the continuity of care is often a problem for patients because the burden of facilitating communication between the three specialties falls on the patient themselves. Treatment options are limited to invasive interventions that can cause debilitating side effects and seriously impact the quality of life. Despite having access to the “best” care, patients with CNS metastasis still have a worse prognosis, disproportionate treatment response, and lower overall survival than patients with metastasis in other organs. The disparities in the quality of care are even more significant among patients with low socioeconomic status as well as patients identified as racial/ethnic minorities—African Americans, American Indians, and Alaskan Natives, Asians, Native Hawaiians/other Pacific Islanders, and Hispanics/LatinosPatients living with CNS metastasis represent a vulnerable cohort, and when medicine and research fail this understudied community, they often turn to other patients and advocates for solutions. Therefore, patient advocates have been charged with leading the effort to address the care gaps and research needs of breast cancer patients who develop CNS metastasis. Though many assume patient advocacy is synonymous with support  patients have become increasingly valuable to the medical and research community, because they offer a unique perspective as experts living with cancer. As the ultimate end users of products developed through research, patients can, and have, helped drive more impactful research that improves survival outcomes.®), the first and only drug approved for BCBM. The Alliance launched the patient-led BCBM Initiative: Marina Kaplan Project, in memory of Marina Pomare Kaplan, with the overarching goal to identify the unmet research needs of patients living with CNS metastasis. The project includes members with representation from industry, research organizations, and individual patients. Nearly one-third of the group is comprised of patients living with brain metastases or LMD. The 17-member scientific advisory board, comprised of a multidisciplinary array of experts in the field of brain metastasis and LMD from breast cancer, advised on the identification of the following gaps in CNS-metastasis research: 1) a poor understanding of the unique brain microenvironment, 2) the absence of sufficient preclinical in vivo animal models that mimic multiple aspects of brain metastasis in a clinical setting, 3) the inability of many anticancer agents to cross either the blood-brain or blood-tumor barrier, 4) the lack of clinically meaningful endpoints that measure survival and quality of life, and 5) the lack of representation of patients with brain metastasis in clinical trials due to restrictive eligibility criteria. Future work addressing each of these gaps will be essential to reduce deaths due to brain metastasis.In 2020, the MBC Alliance patient advocates recognized an opportunity to capitalize on the momentum gained from the approval of tucatinib (TukysaOver the past 20 years, basic research in breast cancer metastasis has led to identifying candidate genes whose expression is predictive of metastasis to specific organs. Prospective studies are warranted to develop assays that detect biomarkers linked to metastatic outcomes. Such improvements would be invaluable for decisions regarding clinical treatment and monitoring.Advances in clinical trial design have allowed subgroup analyses to determine the effectiveness of new therapies of smaller patient subpopulations over the last decade. These analyses have led to the FDA approval of several HER2-targeted treatments that have efficacy for patients with BCBM. Still, much work is needed, particularly to extend outcomes beyond months into years and to consider TNBC. An integrated approach to cancer research that includes the voice of patient advocates will allow us to tackle the remaining challenges while improving the lives and outcomes for patients with breast cancer.


Glioblastoma is one of the most difficult tumor types to treat with conventional therapy options like tumor debulking and chemo- and radiotherapy. Immunotherapeutic agents like oncolytic viruses, immune checkpoint inhibitors, and chimeric antigen receptor T cells have revolutionized cancer therapy, but their success in glioblastoma remains limited and further optimization of immunotherapies is needed. Several oncolytic viruses have demonstrated the ability to infect tumors and trigger anti-tumor immune responses in malignant glioma patients. Leading the pack, oncolytic herpesvirus, first in its class, awaits an approval for treating malignant glioma from MHLW, the federal authority of Japan. Nevertheless, some major hurdles like the blood–brain barrier, the immunosuppressive tumor microenvironment, and tumor heterogeneity can engender suboptimal efficacy in malignant glioma. In this review, we discuss the current status of malignant glioma therapies with a focus on oncolytic viruses in clinical trials. Furthermore, we discuss the obstacles faced by oncolytic viruses in malignant glioma patients and strategies that are being used to overcome these limitations to (1) optimize delivery of oncolytic viruses beyond the blood–brain barrier; (2) trigger inflammatory immune responses in and around tumors; and (3) use multimodal therapies in combination to tackle tumor heterogeneity, with an end goal of optimizing the therapeutic outcome of oncolytic virotherapy. Glioblastoma (GBM) is a grade IV malignant glioma tumor that accounts for the majority 57%) of malignant glioma patients and that remains the most common cause of death due to primary malignant brain tumors in humans ,2. Radio7% of malThe recent success of immunotherapy in clinic, especially with immune checkpoint inhibitors (ICIs) that impede the engagement of programmed cell death 1 (PD-1) or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) with their respective ligand or receptor to boost anti-cancer immunity, has paved a way for these agents to become a part of standard treatment in many cancer types. ICIs have been shown to be effective in patients with an increasingly wide variety of tumors ; howeverOncolytic virotherapy uses replication-competent viruses that can selectively replicate and kill cancer cells ,14,15,16Surgical abscission remains at the core of treatment for malignant glioma along with adjuvant chemoradiation . TemozolA targeted treatment drug, Bevacizumab, has also been approved by the FDA for treating recurrent malignant glioma patients . BevacizUnderstanding the underlying mechanisms behind the development of innate and adaptive resistance to ICIs can help in designing better treatment strategies where ICIs can be used in combination with other therapeutic agents in tumors that are non-responsive to ICI monotherapies. The efficacy of ICIs is dependent on the degree of expression of the target checkpoint receptors on tumor and peritumoral cells, and the heterogeneous nature of malignant glioma tumors can be a major hurdle to success. Systemic immunosuppression , poor imCAR T cell therapy uses autologous T cells engineered to target specific tumor antigens expressed on the surface of tumor cells for tumor eradication. CAR T cell therapies have produced sustained therapeutic effects in refractory hematological cancers, but their success in the treatment of solid tumors has also been limited ,36,37. TImmunomodulation in malignant glioma tumors blocks the activation of immune response pathways important for successful CAR T cell therapy and leads to T cell exhaustion. Therefore, CAR T cell therapy needs further optimization in malignant glioma with improved (1) accessibility to the brain; (2) tumor cell targeting; (3) survivability in immunosuppressive TME; and (4) the ability to proliferate and exert therapeutic effects with minimal immune-based toxicities .n = 11) from patients with recurrent disease showed loss of EGFRvIII expression . F. F85]. FDespite all the hurdles associated with the systemic delivery of OVs, some viruses have shown the ability to effectively cross the BBB to reach and infect tumors in animal models, such as Semliki Forest virus , vacciniMost, if not all, OVs that are in clinical trials for treating malignant glioma are being administered locally to circumvent the barriers associated with systemic delivery of viruses and maximize the virus load in tumors for optimum efficacy. The OVs that are being injected systemically in glioma patients in the ongoing or completed trials include vaccinia virus NCT03294486), reovirus , parvovi486, reovTo summarize, some of the OVs have a natural tropism for neuronal tissue or naturally use immune cells as carriers, enabling them to cross the BBB to infect and kill tumor cells in the CNS compartment; however, most OVs have difficulty in crossing the BBB, upon systemic delivery. The BBB poses as a major hurdle in ensuring efficacious levels of therapeutics are achieved in malignant glioma tumors, including OVs. Carrier cells offer a promising alternative that can help improve the delivery of OVs across the BBB, while ESIA and local CED can potentially maximize the uptake of OVs by tumor cells. Together these strategies can be used to achieve therapeutic levels of OVs in CNS tumors for optimal efficacy.There was an earlier notion that malignant glioma tumors are immunologically privileged because of the isolation from surrounding structures by the BBB ; howeverLocal immunosuppression in malignant glioma tumors is mediated through both the suppression of immune effector cells and the stimulation of immunosuppressive immune cell types. T cell dysfunction in the TME of malignant glioma is mediated via multiple mechanisms that lead to T cell senescence , exhaustSystemic immunosuppression in malignant glioma patients is due to T cell lymphopenia caused by sequestration of T cells in the bone marrow, spleen, and lymphoid organs. Loss of surface expression of S1P1 on T cells, which is critical for egress of T cells from the bone marrow or lymphoid tissues into systemic circulation, is believed to be responsible for severe T cell dysfunction in malignant glioma patients. Tumors located in the CNS, including malignant glioma, disrupt the S1P1–S1P axis gradient to trap T cells in peripheral organs, while causing the contracture of spleen and lymphoid organs .+ and T-bet+ cell infiltration in tumors [As discussed earlier, oncolytic virotherapy can lead to tumor cell lysis, the release of TAAs, the disruption of the immunosuppressive TME, and the induction of innate immune responses in and around tumors. Induction and maintenance of immune responses against tumors is critical for extended therapeutic effects of OVs, post their immune clearance n tumors . Increasn tumors . Oncolytn tumors ,64.As discussed before, malignant glioma tumors secrete immunosuppressive cytokines that help to maintain a tumor protective environment; hence, more immunostimulatory factors in the TME can help to shift the cytokine balance to boost and maintain anti-tumor immune responses. Oncolytic virotherapy uses two main approaches to achieve the higher levels of pro-inflammatory factors in the TME: (1) engineering OVs to express immunostimulatory proteins and (2) the administration of immune stimulators and OVs in combination. Several OVs have been designed to express immunostimulatory cytokines, such as IL-2 ,124,125,The OVs expressing inflammatory cytokines or immune stimulatory factors can effectively turn tumors from “cold” to “hot,” while limiting the toxicities due to systemic administration of pro-inflammatory factors. Furthermore, cancer stem cells that are responsible for the recurrence of disease after chemo-radiation therapy can be effectively killed by OVs . OncolytInfection of host cells by virus can trigger an innate anti-viral inflammatory response, releasing a range of stimulatory cytokines like type I IFN, tumor necrosis factor (TNF), and IL-1, which can be crucial in triggering immune responses against TAAs . AlthougAnother study showed that oncolytic HSV in a murine malignant glioma model enhanced macrophage/microglia infiltration into tumors and polarized them to the pro-inflammatory M1 phenotype that triggered the apoptosis of virus-infected cells, limiting virus spread . MicroglImmune checkpoint receptors are critical to prevent an over-reactive T cell immune response by fine tuning the activation and maintenance of T cell responses. The binding of immune checkpoint receptors expressed on the T cell surface, like PD-1 and CTLA-4, with their ligands sends inhibitory signals to T cells, negatively modulating T cell activity ,154. SevOVs can kickstart inflammatory T cell responses in malignant glioma tumors that can be harnessed with ICIs. The combination of OVs and ICIs is a natural next step and can exert synergistic effects to optimize the therapeutic outcome in malignant glioma patients. Oncolytic adenovirus DNX-2401 in combination with pembrolizumab, an anti-PD1 antibody, is currently in a phase II clinical study (NCT02798406), where the latter is expected to boost the anti-tumor immune response initiated by DNX-2401. There are two other ongoing clinical trials testing the efficacy of nivolumab (anti PD-1 antibody)  in GBM patients. ICIs are being delivered systemically in malignant glioma patients in all current clinical studies and face the obstacle of the BBB, which may reduce their delivery into the CNS compartment. Preclinical studies have shown that the delivery of ICIs across the BBB can be improved by loading them onto nanoparticles resulting in improved overall survival ,159. In P53, Ras, and Rb genes in tumors [Malignant glioma tumors, similar to other solid tumors, show both intra- and inter-tumoral molecular heterogeneity . The levn tumors  or that n tumors ,168,169.The combination of OVs with standard therapeutics like chemo- and radiotherapy can exert a synergistic effect in heterogeneous glioma tumors. Chemotherapeutics induce tumor cell death via different mechanisms including DNA cross-linking , impairment of dsDNA break repair mechanisms (etoposide), and substitution of base pairs (5-FU). Similarly, radiotherapy leads to DNA damage in tumor cells to induce death. DNA repair mechanisms are critical for glioma cells to escape cell death induced by chemotherapeutics and radiation. Oncolytic adenovirus modulates DNA repair mechanisms in tumor cells to enhance tumor sensitization to both chemo and radiotherapies . SeIn the present scenario, a practical way to tackle the tumor heterogeneity and maintain or improve the therapeutic outcome of OVs is to use multimodal combination therapies to achieve synergistic effects. Furthermore, continuous efforts will be needed to develop molecular markers that can be reliably used to identify potential responders to a particular treatment, including OVs. Multifaceted approaches will be crucial when engineering OVs that can exert multimodal effects leading to tumor cell killing to tackle the heterogeneity in glioma tumors.New treatment options will be critical for improving the formidable situation of poor survivability with standard treatment options in malignant glioma patients ,4. Appro