To investigate the prevalence, spectrum, and predictors of alternative diagnoses explaining leg symptoms in patients negative for suspected acute deep venous thrombosis (DVT), which can be detected with whole-leg ultrasound.We retrospectively analyzed a cohort of 789 patients  evaluated with a whole-leg ultrasound examination for suspected acute DVT within one year. All findings in the radiology report were analyzed and electronic chart review was performed to collect clinical information.Ultrasound was negative for acute DVT in 531 patients (67.3%). Among these, alternative diagnoses explaining leg symptoms were seen in 349 patients (65.7%). The most frequent alternative diagnoses were chronic venous insufficiency , followed by lymphedema  and chronic post-thrombotic changes . Patients with alternative diagnoses were older , as well as more likely to present with leg swelling , difference in leg circumference  and redness  than patients without alternative diagnosis. Independent predictors of finding alternative diagnoses on whole-leg ultrasound were older age , leg swelling  and history of previous DVT .Alternative diagnoses explaining leg symptoms can be detected on whole-leg ultrasound in two thirds of patients with no evidence of acute DVT. Our data supports performing a comprehensive ultrasound evaluation beyond the venous system, particularly, in older patients, who present with leg swelling and a past history of DVT. Acute deep venous thrombosis (DVT) is a common condition for which patients seek emergency medical care and is associated with pulmonary embolism as a potentially life-threatening complication. Therefore, accurate diagnosis and prompt initiation of anticoagulant therapy is important to reduce morbidity . ClinicaSome centers choose to perform a limited compression ultrasound examination as the initial test focusing primarily on the femoral and popliteal veins –8. We prIn most cohort studies, the majority of focused ultrasound examinations for DVT in patients with suspected acute DVT are negative (56 to 77%) –15. HoweTherefore, we performed this study to investigate the prevalence, spectrum and predictors of alternative diagnosis explaining leg symptoms among patients evaluated with whole-leg ultrasound for suspected DVT.The study was approved by our institutional review board with waiver of informed consent. The investigation was designed as a retrospective, single-centre cohort study. We included all patients 1) who were examined with whole-leg ultrasound (2) at our institution (3) between January 1 and December 31, 2014 (4) for suspected DVT (5) but no findings suggestive of acute DVT were seen on ultrasound. The year 2014 was chosen because during this year venous ultrasound evaluations were almost exclusively performed by two senior radiologists with extensive experience in vascular ultrasound. We excluded patients with (1) other indications for venous ultrasound and (2) follow-up examinations for established acute DVT diagnosed in the past 3 months  andtargeted ultrasound of symptomatic areas if symptoms are not explained by findings on standard thigh-to-ankle examination.We perform a whole-leg ultrasound of the symptomatic lower extremity in all patients with clinical suspicion of acute DVT in our institution as a combination of color-coded duplex ultrasound, compression ultrasound, and B-mode ultrasound from the groin to the lower leg by using a high-end ultrasound device  and a linear transducer . In the case of suspicion of deep vein thrombosis of the iliac veins, the iliac veins are additionally examined using a convex transducer . The images are stored in the digital image archive . The ultrasound evaluation is performed or supervised by board-certified radiologists with subspecialisation in vascular and interventional radiology. Our protocol includes.Radiology reports of the whole-leg ultrasound examination were retrospectively analyzed for the presence, type, location and presumed etiology of alternative diagnosis.Review of electronic patient charts was performed to record age, gender, presenting symptoms, risk factors, Wells scores and D-Dimer levels.Statistical analysis was performed with GraphPad Prism . Continuous data were presented as median and interquartile range and compared using the nonparametric Mann–Whitney test. Categorical data were displayed as frequencies and proportions and compared between groups using Fisher’s exact test. We performed multiple logistic regression analysis to identify independent predictors of finding an alternative diagnosis in patients without acute DVT on ultrasound. Age and gender were entered into the model as predefined variables. Additionally, we included all variables with significant inter-group differences on univariate analysis. Due to collinearity between both variables, only leg swelling but not measured circumference difference was entered into the model. P values of < 0.05 were regarded as statistically significant.Our final study cohort consisted of 531 of patients with a whole-leg ultrasound negative for acute DVT, of which 269 (50.7%) were women. Patient characteristics are summarized in Table Alternative findings explaining leg symptoms were found on whole-leg ultrasound examination in 349 of 531 patients  and more likely to present with leg swelling , circumference difference  and redness . They were also more likely to have a previous history of DVT . There were no differences in gender, proportion of cancer patients, D-Dimers and Wells score   Cate HoeIn our analysis, older age, leg swelling and previous DVT were independent predictors of finding alternative diagnoses in patients without acute DVT on whole-leg ultrasound. A frequent complication of DVT includes the post-thrombotic syndrome (PTS) with a relatively high number of patients between 20 to 50% within 2 years of DVT diagnosis , 22. CliSince alternative diagnoses are relatively common in patients having a suspicion of acute DVT and these alternative diagnoses have important therapeutic consequences , focusedThis study has some limitations, which include its retrospective single-center nature. Symptomatic patients who presented to a university hospital were included in our analysis. This patient population may be different as compared to patients seen by primary care physicians. No external reference standard was available to confirm the findings. A more comprehensive prospective study is needed to determine the impact of alternative findings on subsequent diagnostic algorithm in patients and the benefits including costs in patient management.In summary, we found that alternative findings explaining leg symptoms can be detected on whole-leg ultrasound in two thirds of patients negative for acute DVT. Therefore, our study supports performing a detailed whole-leg ultrasound evaluation beyond the venous system (including targeted ultrasound of symptomatic areas) when negative for acute DVT. This may be particularly relevant in older patients, who present with leg swelling and who have a previous history of DVT, which were identified as independent predictors in our patient cohort.


Human encephalitis can originate from a variety of different aetiologies, of which infection is the most common one. The diagnostic work-up is specifically challenging in patients with travel history since a broader spectrum of unfamiliar additional infectious agents, e. g. tropical disease pathogens, needs to be considered. Here we present a case of encephalitis of unclear aetiology in a female traveller returning from Africa, who in addition developed an atypical herpes simplex virus (HSV) encephalitis in close temporal relation with high-dose steroid treatment.postmortem brain tissue demonstrated a generalized lymphocytic meningoencephalitis. Immunohistochemical reactions against HSV-1/2 indicated an atypical manifestation of herpesviral encephalitis in brain tissue. Moreover, HSV-1 DNA was detected by a next-generation sequencing (NGS) metagenomics approach. Retrospective analysis of cerebrospinal fluid (CSF) and serum samples revealed HSV-1 DNA only in specimens one day ante mortem.A previously healthy 48-year-old female presented with confusion syndrome and impaired vigilance which had developed during a six-day trip to The Gambia. The condition rapidly worsened to a comatose state. Extensive search for infectious agents including a variety of tropical disease pathogens was unsuccessful. As encephalitic signs persisted despite of calculated antimicrobial and antiviral therapy, high-dose corticosteroids were applied intravenously based on the working diagnosis of an autoimmune encephalitis. The treatment did, however, not improve the patient’s condition. Four days later, bihemispheric signal amplification in the insular and frontobasal cortex was observed on magnetic resonance imaging (MRI). The intracranial pressure rapidly increased and could not be controlled by conservative treatment. The patient died due to tonsillar herniation 21 days after onset of symptoms. Histological examination of This case shows that standard high-dose steroid therapy can contribute to or possibly even trigger fulminant cerebral HSV reactivation in a critically ill patient. Thus, even if extensive laboratory diagnostics including wide-ranging search for infectious pathogens has been performed before and remained without results, continuous re-evaluation of potential differential diagnoses especially regarding opportunistic infections or reactivation of latent infections is of utmost importance, particularly if new symptoms occur. Clarification of the aetiology in a timely manner is crucial regarding therapy and outcome of patients presenting with symptoms of encephalitis , but alsA 48-year-old Caucasian previously healthy female without a history of recreational drug use developed confusion syndrome and haemorrhagic cystitis during a travel to The Gambia Fig. . There sEscherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, Streptococcus pneumoniae, Cryptococcus neoformans/gattii). (II) A group of pathogens was investigated because of the travel history  or (III) because of a potential risk profile that could not be specifically asked . (IV) Moreover, (zoonotic) pathogens which may have been acquired before the journey (Borna disease virus 1 (BoDV-1), Borrelia burgdorferi, Japanese encephalitis virus, Leptospira sp., tick-borne encephalitis virus) or (V) which are rare or very rare pathogens of encephalitis  were examined.During the course of the disease, numerous laboratory examinations were performed in an attempt to identify infectious pathogens potentially responsible for the patient’s encephalitis . Therefore, YEL-AND could not be confirmed.The investigation of a broad spectrum of neuronal autoantibodies , GABARB1/2, GAD, Hu, LGI-1, Ma1/2, NMDAR, Purkinje cells, Ri, Yo) remained without pathological findings and thus, no evidence for an autoimmune-mediated encephalitis existed.On autopsy, neuropathological examination of the brain demonstrated macroscopically oedematous swelling and signs of increased intracranial pressure with sulcal effacement, uncus herniation, and discretely prominent cerebellar tonsils Fig. a. TemporNGS analysis was performed with DNA and RNA isolated from native autopsy tissue of the frontal lobe and cerebellum together with preserved serum from DOS 21. The number of reads that could be assigned to viral specimens was highest in material from the frontal lobe Table .Table 2After HSV DNA and antigen were detected in autopsy specimens of the brain by NGS analysis and immunohistochemical detection, respectively, retrospective conventional single PCR analyses from archived CSF, serum and tissue were performed to confirm these results  followed by autoimmune-mediated encephalitis (21%), while about 40% of the patients remain without a final diagnosis [% followeIn our case, the clinical suspicion of encephalitis quickly emerged and was supported by results of lumbar puncture and EEG. In the initial phase of the disease, diagnostic imaging (CT and MRI) could not reveal a morphological correlate with these findings and the patient’s symptoms. However, a vascular or malignant cause of the disease could be excluded in this way. HSV encephalitis is the most common sporadic form of encephalitis in Western Europe. There is an international consensus to carry out a rapid diagnosis using PCR from the CSF and to administer a empiric therapy with acyclovir. Such measures reduce the lethality of HSV encephalitis from about 70 to 30% .post mortem must indeed have developed towards the end of the clinical course (Table Empiric antibiotic and antiviral treatment was immediately started upon admission to hospital according to the current guidelines of the German Society for Neurology , 7, so tse Table .A travel-associated infectious disease was considered an important differential diagnosis from the very first moment due to the presented neurological symptoms associated with a journey abroad . All relIn addition to the targeted search for pathogens, the ‘extended’ microbiological diagnosis in such a travel-related case must include vaccines applied in a close temporal connection as well as medication already administered on site. A first important aspect of the patient’s travel-related vaccination record is the correctly administered yellow fever vaccination 2 weeks before departure. YEL-AND is a serious side effect after primary vaccination . The freTrichomonas infection was already treated with antibiotics during the stay in The Gambia. Even if the exact active substance was not traceable, a therapy with metronidazole seems to be very likely. Metronidazole-induced encephalopathies described in the literature differ in their typical clinical course from our case [Another important point of the travel history is that haemorrhagic cystitis with proven our case . NeverthWe had expected that the NGS analysis from the brain tissue samples would detect unanticipated or possibly even novel pathogens, which was, however, not the case. Nevertheless, reduced sensitivity remains a weakness of this method. In our case study, for example, an HSV load of 1000 Geq/ml detectable in serum by routine PCR could not generate HSV reads in the NGS investigation of the serum at the same point in time (compare Tables Even today, it is estimated that approximately half of all encephalitis cases remain without identification of a clear cause , 15. DesThe HSV-reactivation presented as an atypical manifestation of HSV-1 encephalitis without the temporally and frontally accentuated haemorrhagic necroses which are typically observed on imaging and autopsy. Such atypical manifestations of HSV-1 encephalitis can be observed in immunosuppressed or critically ill patients –21 and iIn animal models, the hypothesis that naturally occurring stressful situations in the host lead to reactivation of latent herpes viruses due to increased endogenous corticosteroid release was supported early on . The effAlthough it is well known that steroid therapy can cause HSV reactivation in neurons in animal models, only few cases have been reported in the literature in which HSV reactivation and encephalitis coincided with steroid therapy in humans . Of noteIn summary, our case represents the reactivation of HSV-1 infection, likely triggered by steroid therapy in the context of a pre-existing severe encephalitis of unknown cause. In order to not overlook newly occurred and potentially treatable entities, continuous re-evaluation of potential differential diagnoses, especially regarding opportunistic infections or reactivation of latent infections, is of utmost importance.


In tumours, hypoxia—a condition in which the demand for oxygen is higher than its availability—is well known to be associated with reduced sensitivity to radiotherapy and chemotherapy, and with immunosuppression. The consequences of hypoxia on tumour biology and patient outcomes have therefore led to the investigation of strategies that can alleviate hypoxia in cancer cells, with the aim of sensitising cells to treatments. An alternative therapeutic approach involves the design of prodrugs that are activated by hypoxic cells. Increasing evidence indicates that hypoxia is not just clinically significant in adult cancers but also in paediatric cancers. We evaluate relevant methods to assess the levels and extent of hypoxia in childhood cancers, including novel imaging strategies such as oxygen-enhanced magnetic resonance imaging (MRI). Preclinical and clinical evidence largely supports the use of hypoxia-targeting drugs in children, and we describe the critical need to identify robust predictive biomarkers for the use of such drugs in future paediatric clinical trials. Ultimately, a more personalised approach to treatment that includes targeting hypoxic tumour cells might improve outcomes in subgroups of paediatric cancer patients. Hypoxic tumours typically exhibit oxygen tensions of <10 mmHg, whereas in normal tissues, the oxygen tension is in the range of 24–66 mmHg.7 an observation that provided the rationale for the development of hypoxia-targeted treatment strategies.8 The application of such therapies has primarily focused on adult cancers, and any potential benefits have generally been overlooked in paediatric cancers.9 Although the prognosis of paediatric cancers has significantly improved over the past 60 years, cancer remains a major cause of death amongst children.10 There is therefore an urgent need to improve outcomes for childhood cancer by introducing new treatments that are more targeted and reflect key functional differences between individual patients’ tumours.Ultimately, when present, hypoxia leads to poor long-term prognosis in cancer patients,In this review, we describe clinically relevant methods to assess tumour hypoxia in children and outline hypoxia-targeted treatment strategies that could be applied in paediatric cancers, such as reducing the extent of hypoxia to restore sensitivity to treatment, designing hypoxia-activated prodrugs (HAPs) to selectively target hypoxic cancer cells and targeting the adaptive mechanisms downstream of hypoxia that enable tumour cells to survive. The need to include biomarker-led patient selection in clinical trials is also highlighted as a key approach.12 Table 2) with polarographic needles,13 has never been performed in children. Moreover, the limited research into hypoxia biomarkers in paediatric cancers has been to primarily determine their prognostic value, rather than understanding their ability to predict the response to standard or hypoxia-targeted treatments. The following section gives a brief overview of the research available on methods to assess hypoxia in children, and discusses the most relevant techniques to implement in the clinic for paediatric patient stratification.Although numerous hypoxia-targeted treatment strategies have progressed into clinical trials, many of these approaches have not been successfully implemented in the clinic—largely because trial designs have remarkably not included methods to identify and select patients with hypoxic tumours prior to enrolment. Thus, it is necessary to include reliable biomarkers that are predictive of a positive response to hypoxia-targeted treatment strategies in future clinical trials.14 Examples include pimonidazole hydrochloride and the pentafluorinated derivative of etanidazole, EF5.15 Pimonidazole has been used as the standard agent to identify hypoxic areas in tumours in several preclinical studies.14 Furthermore, pimonidazole was shown to be a predictive biomarker of the response to hypoxia-targeted carbogen inhalation (a strategy intended to increase tumour oxygenation) in a Phase 3 trial in adult patients with laryngeal cancer.16 Although there are no known toxicities that might limit the administration of pimonidazole in children, its potential use as a predictive biomarker of response in children has not been investigated, partly because intravenous administration of these compounds prior to tumour biopsy or surgery would be required. However, an advantage of these exogenous markers is that they are activated in the same conditions as some HAPs, being dependent on both hypoxia and one-electron reductase activity,17 which could render them useful as predictive biomarkers of response to HAPs in children.Some of the most commonly used markers of hypoxia in adult cancers are nitroimidazole compounds. In the absence of oxygen, these bioreductive nitroaromatic compounds undergo reduction to form stable adducts with thiol groups in proteins, peptides and amino acids, which can be detected immunohistochemically.19 Differential expression of genes/proteins of the HIF pathway can therefore be used as endogenous markers of hypoxia. The prognostic significance of some of these has been evaluated in paediatric cancers.Tissue hypoxia leads to the activation of HIFs—transcription factors that, in tumours, can mediate the expression of specific genes that orchestrate phenotypic changes, leading to cancer progression and therapy resistance.20 By contrast, HIF-1α is an unfavourable prognostic factor in bone cancers, including osteosarcomas.21 Various paediatric cancers have elevated levels of vascular endothelial growth factor (VEGF), a HIF downstream target, which are associated with worse outcomes.9 However, levels of VEGF do not correlate with neuroblastoma stage, or its genotype or phenotype.20 Another downstream target of HIF, glucose transporter 1 (GLUT1), is more frequently expressed in neuroblastic tumours (including neuroblastomas) with poor prognosis and unfavourable pathology.22 In a separate study, a small cohort of neuroblastoma specimens was evaluated for levels of the HIF downstream targets carbonic anhydrase IX (CAIX) and phosphoglycerate kinase 1 (PGK1); although higher expression of PGK1 has been correlated with strong metastatic behaviour,23 this study concluded that CAIX was a stronger negative prognostic factor for survival.24 In a small cohort, high levels of CAIX expression in osteosarcoma tumour samples were also shown to be linked with a marginally worse prognosis than low or no CAIX expression, suggesting the potential use of CAIX as a biomarker of high-risk osteosarcoma.25 Lastly, lysyl oxidase is another downstream target of HIF that has been shown to be expressed in up to two-thirds of samples from a cohort of patients with retinoblastoma.26 Surprisingly, retinoblastoma cell lines were more sensitive to treatment with common chemotherapeutic agents under hypoxic conditions.26 This demonstrates the variable significance of these endogenous hypoxia markers in different cancer types, highlighting the importance of investigating them in individual cancers.In neuroblastoma, the most common extracranial solid tumour in infancy, higher expression of the HIF-1 subunit α (HIF-1α) correlates with low tumour grade and favourable patient prognosis, whilst HIF-2α expression correlates with high tumour stage and unfavourable prognosis.27 Further studies must be carried out to understand the potential role of these markers as predictors of response not only to existing treatments, but also to novel hypoxia-targeted treatments.Although establishing prognostic significance can support the rationale for targeting hypoxic cells in childhood cancers, it is not enough on its own to ensure that novel agents will be effective in patients. HIF-1α, VEGF, CAIX and GLUT1 were collectively assessed immunohistochemically in tumour tissue microarrays of a small cohort of patients with rhabdomyosarcoma to predict the response to neoadjuvant chemotherapy. The study revealed that the co-expression of three or more of these markers was an independent predictive factor of poor response to chemotherapy.28 however, how hypoxia specifically influences the release of osteopontin into the bloodstream remains elusive. Osteopontin levels have been shown to correlate with direct O2 measurements, predicting disease progression and response to treatment in some adult cancers.30 The potential use of osteopontin as a biomarker has been demonstrated in childhood cancers, such as acute lymphoblastic leukaemia (ALL), malignant gliomas31 and osteosarcoma,32 although further investigation is required for clinical use.The endogenous hypoxia marker osteopontin can be detected and quantified in blood samples without the need to carry out a tumour biopsy, enabling the longitudinal assessment of the overall tumour hypoxia burden. Osteopontin is a tumour-associated glycoprotein secreted into the blood plasma that plays a role in several physiological processes, such as tissue remodelling and inflammation;33 A 15-gene signature successfully predicted the clinical outcome and response to hypoxia-targeted therapy in combination with radiotherapy in head and neck squamous cell carcinoma (HNSCC), serving as a prognostic and predictive marker.34 This signature was further validated in other cancer types and shown to be the most robust hypoxia gene signature of all those assessed.35 Another hypoxia-related 28-gene signature was validated in prostate cancer as a prognostic and predictive biomarker for assessing the benefit of hypoxia-modifying therapy.36 A 24-gene hypoxia signature has been defined in bladder cancer patients37 and another in soft-tissue sarcoma patients.38Several attempts have been made to identify a collection of genes whose expression levels can reflect tumour hypoxia and be used to identify any correlations with disease progression or response to therapy—a hypoxia gene-expression signature. Approximately 30 hypoxia gene signatures have been identified for adult cancers.39 Currently, hypoxia gene signatures are derived from single biopsy samples, and for this method to be clinically relevant in children, it is essential that a single biopsy sample is representative of the entire tumour.The reliability of a single biopsy sample to reflect the hypoxic status of a tumour was compared with multiple biopsy samples using gene signatures, including three of the signatures mentioned above, or single gene markers of hypoxia. The study demonstrated the ability of a gene signature from a single biopsy sample to correctly classify a tumour as more or less hypoxic, although small variability between biopsy samples remains unavoidable.40 thus representing a potential novel hypoxia gene signature for a paediatric cancer.The outlook for identifying and using hypoxia gene signatures in paediatric cancers is encouraging, although data are currently limited. In 2016, a set of nine genes identified in neuroblastoma tumour samples and hypoxic neuroblastoma cell lines was shown to be associated with aggressive neuroblastomas,41 There is a pressing need for imaging biomarkers not only to identify patients with hypoxic tumours, but also to map the degree and distribution of hypoxia to optimise radiotherapy planning and to assess the effects of hypoxia-modifying approaches for therapeutic gain.43Non-invasive imaging methods to repeatedly and rapidly quantify the extent and heterogeneity of hypoxia within an individual tumour in vivo would offer a clinical benefit in personalised treatment planning.44Magnetic resonance imaging (MRI) provides a non-invasive approach for monitoring cancer. MRI exploits the magnetic moment of protons in water within the tissue to create high-resolution anatomical images with exquisite soft-tissue contrast. As non-ionising radiation is used, MRI lends itself well to repeat investigations. Image acquisition can be sensitised to numerous independent contrast mechanisms, from which parametric maps can be calculated and used to provide additional information on tumour structure and function. Such multiparametric MRI strategies can thus be used to evaluate intratumoural heterogeneity and its relationship to tumour progression and treatment response.46 One approach, intrinsic susceptibility MRI (IS-MRI), exploits the paramagnetic properties  of deoxyhaemoglobin in erythrocytes to create image contrast. Deoxyhaemoglobin therefore acts as an intrinsic, blood oxygenation-level-dependent contrast agent . IS-MRI investigations quantify native tumour R2* and/or the change in R2* (ΔR2*) induced by respiratory challenge with a high-oxygen content gas. Hyperoxia-induced decreases in R2*, which reflect changes in blood oxygenation due to a change in haemoglobin saturation, have been shown to be associated with improved tumour oxygenation.48 A series of preclinical IS-MRI studies performed in two separate laboratories using syngeneic rat rhabdomyosarcoma models have shown that this tumour type exhibits a relatively high native R2* and a marked hyperoxia-induced reduction in R2*, consistent with a hypoxic phenotype.50 These studies also highlight the potential of IS-MRI to provide predictive imaging biomarkers of rhabdomyosarcoma response to radiotherapy. Investigation of tumours arising in transgenic mouse models of neuroblastoma revealed extremely high native R2* values and marked hyperoxia-induced ΔR2*, but with negligible evidence of hypoxia assessed histologically.51 The IS-MRI data are consistent with aberrant haemodynamic vasculature typically ascribed to this paediatric cancer. Collectively these preclinical studies highlight that the relationship of native R2* to tissue hypoxia can vary according to the underlying histology.46 This is important because the underlying histology influences the level of hypoxia in a tumour.Several MRI techniques are being actively investigated for delivering translational imaging biomarkers of hypoxia.1 (s−1)—caused by excess oxygen molecules dissolved in blood plasma and interstitial fluid.52 In oxygenated tissues, hyperoxia induces a change in the signal intensity,53 whereas in hypoxic tissue, oxygen molecules that have been inhaled preferentially bind to deoxygenated haemoglobin, rather than dissolving in blood plasma and interstitial fluid, resulting in unchanged signal intensities. Comparison with image-aligned tissue sections stained for pimonidazole showed that the perfused tumour areas refractory to hyperoxia-induced changes were hypoxic.53 Preclinical and clinical studies have demonstrated that the volume of oxygen-refractory voxels can be used to map and quantify hypoxic fractions across a range of tumour types.57 OE-MRI revealed extensive tumour subregions that were refractory to oxygen-induced signal intensity changes in a syngeneic rat rhabdomyosarcoma model, again consistent with the presence of hypoxia.50 Preliminary OE-MRI investigations in RH41 rhabdomyosarcoma xenografts also suggest a strong hypoxia signature in this tumour type , relies on quantifying hyperoxia-induced signal changes—the longitudinal MRI relaxation rate R58 Whilst the application of MRI protocols in children can be challenging due to the logistical challenges involved, such as having to sedate/anaesthetise younger patients to undergo the exam, rapid mapping of tumour hypoxia with IS-MRI and OE-MRI, which both rely on endogenous contrast mechanisms, is totally non-invasive and relatively straightforward to incorporate into existing clinical imaging protocols. We predict that within the next 5–10 years, this is likely to enhance imaging-embedded investigations associated with clinical interventions that may ultimately improve the outcome for paediatric patients.MRI is used routinely in healthcare, and both IS-MRI and OE-MRI have been implemented on standard clinical scanners and used to assess tumour hypoxia in adult patients.59 Many early-phase clinical trials in children rely on successful adult Phase 3 trials; however, it is important to consider—from a therapeutic perspective—that differences exist in the aetiology between adult and paediatric cancers.60 Nevertheless, hypoxia has been shown to be important in several paediatric cancers. As outlined above, the presence of endogenous hypoxia markers has a negative impact upon paediatric cancer prognosis. Hypoxia is also likely to be a treatment-limiting factor in the most prevalent paediatric cancers, such as leukaemia, brain cancer and sarcomas: for instance, chemoresistance was demonstrated to be promoted by hypoxic conditions in ALL cell lines via the modulation of cell death signalling pathways.61 Medulloblastoma cell lines under hypoxic conditions have a significant reduction in therapy-related DNA damage after treatment with etoposide and radiotherapy.62 Similarly, rhabdomyosarcoma and Ewing’s sarcoma cell lines develop resistance to treatment-induced apoptosis in hypoxic conditions.63 Therefore, the inclusion of hypoxia-targeted approaches in treatment regimens has the potential to benefit paediatric cancer cases.Hypoxia is a potentially targetable characteristic of most locally advanced solid tumours.8 These strategies are summarised in Fig. 64 whereas in solid tumours, median oxygen levels can vary between 0.366 and 4.2%.67 As areas of pathological hypoxia are heterogeneously distributed throughout tumours13 between areas of physiological oxygenation, it might be necessary to combine hypoxia-targeting drugs with existing therapies—chemotherapy, radiotherapy and immunotherapy—to effectively treat all regions.18To date, three main hypoxia-targeted strategies have been developed. The first involves increasing the level of oxygen in the tumour microenvironment (TME). Alternatively, hypoxia can be exploited so that hypoxic cancer cells are selectively targeted. Finally, the adaptive mechanisms that enable tumour cells to survive in hypoxia can be targeted.71 Hypoxia features in the microenvironment of bone marrow, and increased HIF-1α expression and the acquisition of a glycolytic phenotype  was demonstrated in co-cultures of leukaemic cells with bone marrow-derived mesenchymal stem cells under hypoxic conditions.68 Medulloblastoma cell lines upregulate HIFs under hypoxic conditions, and a decrease in cellular proliferation was observed in response to knockdown of the HIF1A gene.69 In studies of cells derived from primary Ewing’s sarcoma family tumours, the hypoxic microenvironment was shown to upregulate expression of the EWS–FLI1 fusion protein70 and to increase cellular invasion in vitro.71 Thus, the interactions between the various components of a tumour influence the tumour’s ability to proliferate and metastasise, and its response to therapy.59 Treatments that improve oxygen availability to hypoxic cancer cells in the TME aim to sensitise tumours to existing therapies.Tumour cells evolve in parallel with various components of the TME, including the extracellular matrix and cells of the vascular, stroma and immune systems. The influence of hypoxia on the TME of paediatric cancers is documented in multiple studies.72 Later studies conducted in irradiated, tumour-bearing mice, demonstrated greater tumour regression in mice inhaling 100% oxygen rather than ambient air.73 Thomlinson and Gray were the first to infer the presence of hypoxic cells in human lung cancer sections and comment on their implications for radiotherapy.74The first correlation between the availability of oxygen and radiosensitivity was discovered in the early twentieth century whereby greater inhibition of root growth during germination was observed with aerobic seeds compared with anaerobic seeds after irradiation.75The earliest attempts to increase tumour oxygenation during radiotherapy included the use of hyperbaric oxygen, blood transfusions and administration of erythropoietin (to stimulate the generation of red blood cells from stem cells). These therapies never became standard-of-care treatment, however, due to conflicting efficacies in clinical trials.16 Improved 5-year overall survival (OS) rates (50% compared with 39%) were also reported in adult patients with bladder cancer.76 In both cases, carbogen was co-administered with nicotinamide, which reduces fluctuations of blood flow.75 Retrospective studies using hypoxia gene signatures for both trials confirmed that hypoxic tumours responded more optimally than normoxic tumours to hypoxia-modifying treatment.77 Despite some reports of breathing discomfort alongside carbogen inhalation, compliance in adults is high;78 in children, carbogen inhalation per se is reported to be safe and feasible without any significant compliance issues.81 It is, however, foreseeable that this therapy would be more suitable for older children, or toddlers and babies that have already been sedated for radiotherapy. In a clinical trial that combined carbogen inhalation with radiotherapy for the treatment of high-grade paediatric gliomas, no increase in survival over radiotherapy alone was noted.82 It is important to note, however, that tumour hypoxia was not evaluated in this small trial, and so the need to select for compliant patients with hypoxic tumours for paediatric cancer clinical trials remains untested.A further method to increase tumour oxygenation is the inhalation of carbogen—usually a mix of 5% carbon dioxide and 95% oxygen gas—during radiation treatment. Carbogen inhalation proved its efficacy in Phase 3 trials, significantly improving 5-year regional control  in adult patients with HNSCC.83 Mathematical modelling has shown that decreasing oxygen consumption in well-perfused areas is more effective at increasing oxygen availability in hypoxic areas than attempts to increase oxygen supply in these areas.84 The antidiabetic drug metformin alleviates hypoxia by inhibiting oxidative phosphorylation through inhibition of mitochondrial complex I, thereby reducing the oxygen consumption rate (OCR) in cancer cells.85 A 2018 Phase 1 trial of advanced HNSCC evaluating the addition of metformin to standard cisplatin and radiotherapy showed promising results  with 2-year OS rates and progression-free survival (PFS) rates of 90 and 84%, respectively. A Phase 2 trial for this combination in adult cervical cancer (NCT02394652) is currently at the recruitment stage.Decreasing the oxygen consumption of perihypoxic cells in tumours is another approach to modify the level of oxygen availability.86 Conversely, the inhibitory effects of metformin on tumour growth were reduced by hypoxia in childhood sarcoma xenografts.87 The disparity in results may be due to the inherent differences between hepatomas and sarcomas and/or the variabilities in experimental details, including drug concentrations and methods used to induce hypoxia. The pleiotropic effects of metformin on cancer cells are also apparent in adults, with inconsistent outcomes in adult clinical trials, too.88 Nonetheless, metformin is currently being tested in a Phase 1 clinical trial that aims to evaluate escalating doses of the agent in combination with a backbone therapy of vincristine, irinotecan and temozolomide in children with recurrent and refractory solid and brain tumours (NCT01528046).90Preclinical evidence supporting the use of metformin in childhood cancers is variable. Metformin successfully restored chemosensitivity to cisplatin in hepatoblastoma cells under hypoxia.91 Metformin was also tested; however, the dose required to achieve a similar reduction in OCR is not achievable in patients. Atovaquone can therefore be added to standard-of-care treatment without additional severe toxicities in patients.91 These results have prompted a clinical trial (NCT02628080) for non-small- cell lung cancer in which immunohistochemical, serological and imaging of hypoxia biomarkers are evaluated in addition to clinical outcomes.91Atovaquone is an antimalarial drug found to reduce OCR—to an even greater extent than metformin—through the inhibition of mitochondrial complex III. Clinically achievable doses of atovaquone are capable of reducing OCR in vitro and alleviating tumour hypoxia in vivo, and consequently increase the radiosensitivity of various cancer types.Pneumocystis carinii.92 Repurposing safe, commonly prescribed drugs is a superficially favourable approach, as the limitations of testing new agents in children exceed those for adults; known safety and toxicity profiles shorten the timeline and reduce the number of patients required—already scarce to start with—to obtain approval for a novel indication in children.94 Ethically, these patients must receive the highest standard of care and some patients/parents are not willing to accept the safety risks involved with Phase 1 testing.93 Together with the lack of commercial interest to develop new drugs with the sole indication of treating rare paediatric cancers,94 these factors support repurposing atovaquone in children, although preclinical evidence must be generated to support this combination before clinical trials commence.Atovaquone is successfully used in children with leukaemia as a prophylactic approach against 15 hence the name hypoxia-activated prodrugs (HAPs). HAPs can be divided into five main subgroups: nitroaromatic compounds , aromatic N-oxides , aliphatic N-oxides, quinones and metal complexes. Of these subgroups, nitroaromatic compounds and aromatic N-oxides have progressed the furthest in clinical development and have been preclinically tested.Oxygen concentrations are frequently markedly lower in malignant tissues compared with their non-malignant surroundings, rendering hypoxia a targetable feature in tumours. Bioreductive prodrugs have been developed to exploit the endogenous expression of human cellular oxidoreductases in hypoxic tumour cells, such that only in the absence of oxygen, can oxidoreductases reduce the prodrug and convert it into an unstable prodrug radical. This in turn can then be further reduced or fragmented into a toxic metabolite. Hence, the conversion of the prodrug into its toxic metabolite is activated under conditions of hypoxia, but inactivated in the presence of oxygen:15 For example, an active bystander effect was observed using PR-104 in a three-dimensional spheroid model combined with mathematical modelling.95 Single-agent activity of PR-104 in mouse xenograft models96 and activation in oxic cancer cells by human aldo–keto reductase 1C397 have both been demonstrated. Single-agent efficacy of evofosfamide is due to very high plasma concentrations of the drug, achievable in patients. This provides significant bioreductive activation despite partial inhibition by oxygen.98 Evofosfamide has single-agent efficacy in vitro, and in mouse xenograft tumour models of adult nasopharyngeal carcinomas,99 as well as in patient-derived xenograft models of HNSCC.100As a single-agent therapy, HAPs must target both the hypoxic and normoxic components of tumours. This can be achieved through the active bystander effect, whereby the active metabolite of the HAP is not only generated in hypoxic areas, but also diffuses into neighbouring normoxic cells.64 Predicting off-target toxicity will therefore be fundamental for the implementation of these agents. The spatially resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) model is potentially a valuable tool for predicting off-target toxicity.101Although single-agent activity might be desirable, the activation of prodrugs by intermediate levels of oxygen could limit their clinical use due to the potential risk of toxicities in healthy tissues undergoing physiological hypoxia, such as in the liver and brain.102 The first available nitro-based HAPs, such as misonidazole, proved to be clinically disappointing, however, as undesirable side effects limited the dose.103 Nimorazole was soon demonstrated to be less toxic and to have similar radiosensitising properties at clinically achievable doses.104 Nimorazole significantly improved the efficacy of radiotherapy in adult HNSCC without major side effects, leading to its routine clinical use in Denmark.105 Consequently, a UK-based Phase 3 clinical trial was set up to assess the benefit of using nimorazole during radiation treatment of patients with HNSCC, the results of which are yet to be published.106 These studies suggest that nimorazole could be a potential therapy for paediatric cancers, although this notion remains untested to date. Although tolerance in adults due to nausea and vomiting is an issue, there are no obvious concerns for the use of nimorazole in children and it is less toxic than similar compounds, such as misonidazole and etanidazole.Early members of the class of nitroaromatic compounds were shown to mimic the radiosensitisation caused by oxygen in non-hypoxic tissues, and were therefore originally developed to be radiosensitisers, so-called oxygen mimetics.102 Both drug classes constitute HAPs as they depend on the absence of oxygen for activation by endogenously expressed oxidoreductases. However, cytotoxic DNA-targeting HAPs cause DNA damage, whereas oxygen mimetics merely stabilise existing radiotherapy-induced DNA damage, as oxygen does.107 The different modes of action of HAPs are illustrated in Fig. 108 A Phase 3 trial testing this same combination in HNSCC patients also failed to show any improvement in OS.109 However, neither of these trials selected patients with hypoxic tumours for treatment with tirapazamine, and it was suggested in both cases that biomarker-led patient selection would improve the results. Evofosfamide was shown in 2019 to sensitise oesophageal cancers to radiotherapy without increased levels of toxicity in vivo.110 Similar results were obtained when this combination was tested in a syngeneic rat rhabdomyosarcoma model. The authors noted that the efficacy of treatment was dependent on the tumour oxygenation status, which was evaluated using [18F]HX4 hypoxia PET imaging.111 Again, including hypoxia biomarkers in clinical trials could enhance the efficacy of these treatments.The development of cytotoxic DNA-targeting bioreductive agents  soon followed the development of oxygen mimetics.112 The combination of evofosfamide with topotecan, a topoisomerase inhibitor, was investigated in cell line models of neuroblastoma and rhabdomyosarcoma. In contrast to single agents, the combination treatment led to complete tumour regression and improved response in the neuroblastoma and rhabdomyosarcoma models, respectively. Moreover, the combined treatment was shown to induce tumour cell apoptosis in both hypoxic and non-hypoxic regions.113The majority of clinical trials of cytotoxic HAPs have involved combination with chemotherapeutic agents. In this approach, non-hypoxic cells within a tumour are expected to be primarily targeted by the cytotoxic activity of chemotherapeutic drugs, whilst hypoxic cells are targeted by HAPs, thereby treating both cellular populations of the tumour.114 and has been investigated as a promising drug candidate for paediatric cancers. Unfortunately, however, the effectiveness of tirapazamine in treating paediatric rhabdomyosarcoma could not be demonstrated in the absence of predictive patient stratification.115 Similarly, in adults, large, randomised Phase 3 trials failed to achieve positive results, despite promising Phase 1 and 2 trials for tirapazamine and evofosfamide.12 For example, the TH CR-406/SARC021 trial in patients with locally advanced, unresectable or metastatic soft-tissue sarcoma, based on promising Phase 2 trial results,116 failed to show any benefit from the addition of evofosfamide to doxorubicin.117 The level of tumour hypoxia prior to the administration of hypoxia-targeted therapies was not evaluated in any of these trials. Although the combination of HAPs with chemotherapy remains a promising therapy for paediatric cancers, again, reliable biomarkers must be identified to effectively predict the response to HAPs and to identify which patients are most likely to benefit from these therapies.Tirapazamine is clinically safe in children,118 HIF-mediated resistance to chemotherapy is linked to the induction of autophagy, defective apoptosis, reduction of DNA damage and overexpression of drug efflux proteins.76 In hypoxic conditions, resistance to radiotherapy is primarily due to the lack of oxygen to stabilise radiation-induced free radicals.119 Additionally, increased HIF activity has a protective effect on the tumour vasculature.120 Higher HIF-1 expression levels after radiation lead to the secretion of VEGF, which inhibits apoptosis in endothelial cells, thereby reducing the vascular damage normally caused by radiation.121 The discovery of this key regulatory pathway has led to several attempts to inhibit HIF and its downstream targets.122The activation of HIFs in hypoxic tissues promotes angiogenesis, metabolic reprogramming and a stem-cell-like phenotype, which collectively increases tumour invasion and metastasis.124 HIF-1 inhibitors such as PX-478 and NSC-134754 were also included in adult Phase 1 clinical trials,9 but as yet, have not been introduced in the paediatric cancer setting. Selectively inhibiting HIF has proved to be a challenge, and no small-molecule HIF-1 inhibitors have so far been clinically approved, most likely because they lack the desired specificity.125Geldanamycin, a small molecule that indirectly inhibits HIF activity, showed promising results in paediatric Phase 1 trials. However, dose-limiting toxicities were observed in one of the two trials.126 and is now emerging as a potential target. A Phase 3 trial for adult clear-cell renal cell carcinoma is planned with the second-generation HIF-2α inhibitor, PT2977.127 A first-generation HIF-2α inhibitor, PT2385, was unable to inhibit downstream signalling and had no effect on the response to chemotherapy when tested in paediatric neuroblastoma patient-derived xenografts, implying that this may not be an appropriate target in neuroblastoma.128 Furthermore, another study found that HIF-2ɑ demonstrates tumour-suppressive properties in neuroblastoma tumours treated with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) and retinoic acid.129HIF-2α has been described to have an important role in tumour progression,130 Combining the inhibition of histone methyltransferase enhancer of zeste homologue 2 (EZH2) with HIF inhibition has also been proposed as a potential new treatment strategy for paediatric high-grade gliomas.131 Acriflavine, a safe, small molecule with HIF-inhibiting properties, was shown to downregulate HIF-mediated pathways and induce cell death in both in vitro and in vivo models of glioma.132Nonetheless, interest in HIF inhibition as a therapeutic strategy for paediatric cancer continues, particularly for paediatric brain tumours. Blocking HIF-1α transcriptional activity with 2-methoxyestradiol increased the cytotoxic activity of cyclophosphamide and ifosfamide in medulloblastoma cells.121 and PX-478133 can radiosensitise tumours in mouse xenografts, less toxic and more effective drug candidates are required to progress to clinical testing.The combination of HIF inhibition plus radiotherapy has been preclinically evaluated. Although the HIF inhibitors YC-1Together, these findings suggest that HIF inhibitors might still have potential for paediatric cancer treatment.18 GLUT1 and CAIX are proteins located on the cell membrane with a functional role in glucose uptake and pH regulation, respectively. They increase glycolytic metabolism and enhance cell survival in low oxygen environments.134 These HIF downstream target genes can be inhibited, and have been investigated for therapeutic benefit to varying extents.VEGF plays a central role in regulating angiogenesis in hypoxic tumours, whilst GLUT1 and CAIX contribute to adaptations in hypoxic cancer cell metabolism.135 The radiation–bevacizumab combination is effective due to the temporary ‘normalising’ effect of VEGF inhibitors on the tumour vasculature: immediately after treatment, tumour perfusion and oxygen supply are temporarily restored due to the emergence of stable and more mature blood vessels, and pruning of immature and chaotic vessels conferring greater radiation-induced DNA damage.136 However, this restoration effect is followed by blood vessel regression, eventually leading to poor perfusion and a decrease in tumour oxygenation in tumours, during which time radiotherapy is less effective.20 The success of this combination relies on the development of suitable imaging and blood-based biomarkers to determine scheduling regimens as well as selecting suitable patients for treatment.136 Notably, combining VEGF inhibition with chemoradiotherapy in glioblastoma had no effect on OS in two Phase 3 trials, but resulted in more adverse effects. Patients in these studies were not pre-selected based on predictive markers.138The combination of radiotherapy plus the anti-VEGF antibody bevacizumab has been investigated in a number of adult clinical trials.139 It demonstrated tolerability and safety in children in a Phase 1 trial in paediatric patients with refractory solid tumours140 and has since entered into Phase 2 trials. The Phase 2 BERNIE study in metastatic paediatric soft-tissue sarcomas concluded that the addition of bevacizumab to chemotherapy brought about no significant improvement in event-free survival.141 Similarly, bevacizumab in combination with irinotecan in paediatric malignant glioma and diffuse brainstem glioma failed to improve outcomes in children.142 However, in recurrent low-grade gliomas, the same combination produced sustained disease control in some children.143 The blood vessel regression caused by anti-angiogenic therapies can reduce perfusion levels, thereby limiting optimal drug delivery and further increasing tumour hypoxia.144 More research is required to determine which paediatric cancers might benefit from this combination.Bevacizumab is used in combination with chemotherapy or as a single agent in selected adult cancers.145 SLC-0111 is being tested in a Phase 1/2 trial in combination with gemcitabine for pancreatic cancer (NCT03450018), and a Phase 1 trial for the treatment of advanced metastatic solid tumours was completed successfully.146 The potential of this treatment strategy is in the developmental stages, but considering that CAIX expression correlates with worse prognosis in certain childhood cancers,25 this approach could similarly be applied for treatment of paediatric cancers. Lastly, GLUT1 inhibitors, such as BAY-876147 and WBZ117, successfully restored radiosensitivity in radioresistant breast cancer cells.148Over the past 5 years or so, inhibitors of CAIX, such as SLC-0111, which sensitises several cancer cells lines to conventional chemotherapy, have been developed.150 The resistance of various tumours to immunotherapeutic drugs could therefore be attributed to hypoxia. However, to date, the effect of tumour hypoxia on treatment response is only being evaluated in one immunotherapy trial (NCT03003637), in which adult patients are treated with nivolumab, a programmed cell death 1 (PD-1) inhibitor, in combination or not with ipilimumab, a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitor, in addition to standard of care.151Increasing evidence supports the notion that hypoxia is immunosuppressive, decreasing the tumour innate and adaptive immune response.152 An ongoing trial (NCT03098160) is investigating the combination of evofosfamide plus ipilimumab in several adult cancers.153 In a melanoma mouse model study, tumour growth was delayed, and survival was improved after combination treatment with SLC-0111 plus immune-checkpoint blockade.154In an attempt to reverse the effects of hypoxia on anti-tumour immunity, the combination of immunotherapy plus hypoxia-targeted agents has been investigated. The benefit of targeting hypoxic cells with evofosfamide in combination with CTLA-4 and PD-1 checkpoint blockade was demonstrated in a transgenic adenocarcinoma mouse prostate model; this treatment resulted in a 30–50% increase in OS compared with single-agent treatment, with elevated levels of infiltrating T lymphocytes in hypoxic areas of tumours.155 Whereas CAR T-cell therapy has produced some impressive results in this subset of patients, immune-checkpoint inhibitors have been less successful, possibly owing to the lack of neoantigens in paediatric cancers:155 as mentioned above, differences in the aetiology of adult and paediatric cancers exist—one example being the environmental induction of a high mutational burden in adult cancers that is largely absent from paediatric tumours.156Immune-checkpoint inhibitors and chimaeric antigen receptor (CAR) T-cell therapy have been approved for the treatment of a subset of paediatric cancers.157 although generally, fewer patients present with toxicities than those receiving more conventional therapies. However, the risk of severe and potentially fatal side effects remains and tends to be greater in children than in adults. For example, paediatric ALL patients appear to suffer from cytokine-release syndrome and neurological toxicities more frequently than adult ALL patients following treatment with CD19-targeted CAR T-cell therapy.158Immunotherapy for paediatric cancer patients is not without challenges159 Alleviating hypoxia in paediatric cancers could therefore potentially create the necessary pro-inflammatory environment for immunotherapies to be effective. The combination of hypoxia-targeted treatments and immunotherapy remains untested in paediatric cancers, both preclinically and in clinical trials, as research in this field is in its infancy. Paediatric clinical trials will probably only follow successful adult clinical trials, and at this point, it is key that the level of hypoxia is evaluated as a potential prognostic or even predictive biomarker of response for immunotherapy treatment or combined hypoxia-targeted/immunotherapy approach, respectively.Furthermore, hypoxia plays a major role in creating an immunosuppressive environment by stimulating the release of immunosuppressive metabolites.160Adult cancer patients have benefited from being involved in clinical trials of hypoxia-targeted therapies; however, these treatments have largely been given in the absence of stratification by hypoxic status prior to enrolment—meaning little progress has been made, and these treatments are not commonly used in clinical practice. Importantly, as highlighted by Yang et al., studies that have prospectively or retrospectively stratified patients on the basis of hypoxia gene signatures have demonstrated that hypoxia-modifying treatments are more effective the more hypoxic the tumour is.To identify paediatric cancer patients that will benefit from hypoxia-targeted therapies, reliable biomarkers of hypoxia for children must first be developed. The benefit of novel hypoxia assessment techniques in children must outweigh the potential invasive nature of the technique. Three methods appear to be the easiest to integrate into routine diagnostic procedures. The endogenous marker osteopontin can be measured in patient blood samples, despite its limited specificity as a hypoxia marker. Furthermore, once validated for paediatric cancers, hypoxia gene signatures could be relatively easily evaluated in standard tumour biopsy samples. Novel IS- or OE-MRI imaging techniques, already implemented on standard clinical scanners, would allow for the longitudinal, minimally invasive assessment of tumour hypoxia levels without added radiation.If patients can be systematically evaluated for the presence of hypoxia in their tumours, a few therapies might be translatable to children. Nimorazole, in combination with nicotinamide, has shown its immense therapeutic value as a radiosensitiser in HNSCC. Evofosfamide has been widely investigated in paediatric cancers as a HAP and, with biomarker-led clinical trials, it is likely to prove its clinical use in both adults and children. Finally, atovaquone, also a potential radiosensitiser, could be introduced relatively efficiently following its proven safety track record in children.To protect children from unnecessary experimental treatments, it is essential that future clinical trials in children are based on sound rationale for the combination of hypoxia-targeted agents with existing treatments. This must be supported by robust preclinical data and inclusion of the least invasive and most informative techniques to stratify patient tumours according to their level of hypoxia.


Psingle), two (Pmix) or three  cognitive tasks and compared the pattern of modulations between participants with ADHD (n-89), unaffected siblings (n = 93) and controls . Participants with ADHD had significantly fewer Pall connections (modulated regardless of task), but significantly more task-specific (Psingle) connectivity modulations than the other groups. The amplitude of these Psingle modulations was significantly higher in ADHD. Unaffected siblings showed a similar degree of Pall connectivity modulation as controls but a similar degree of Psingle connectivity modulation as ADHD probands. Pall connections were strongly reproducible at the individual level in controls, but showed marked heterogeneity in both participants with ADHD and unaffected siblings. The pattern of reduced task-generic and increased task-specific connectivity modulations in ADHD may be interpreted as reflecting a less efficient functional brain architecture due to a reduction in the ability to generalise processing pathways across multiple cognitive domains. The higher amplitude of unique task-specific connectivity modulations in ADHD may index a more “effortful” coping strategy. Unaffected siblings displayed a task connectivity profile in between that of controls and ADHD probands, supporting an endophenotype view. Our approach provides a new perspective on the core neural underpinnings of ADHD.Attention-deficit/hyperactivity disorder (ADHD) is associated with altered functioning in multiple cognitive domains and neural networks. This paper offers an overarching biological perspective across these. We applied a novel strategy that extracts functional connectivity modulations in the brain across one ( Multiple cognitive theories have been proposed to explain the underlying core deficits of the disorder, including a dysfunction in state and arousal regulation2, response inhibition3, broader executive functioning4, motivation5 and/or delay aversion4. Functional imaging studies building on these cognitive explanations have investigated the neural underpinnings of ADHD, but have revealed a heterogeneous pattern of altered neuronal function spread across the brain8. This fragmented pattern of findings asks for new integrated approaches that provide an overarching perspective on the functional architecture of the ADHD brain across cognitive domains.Attention-deficit/hyperactivity disorder (ADHD) is a mostly early onset neurodevelopmental disorder characterised by symptoms of inattention and/or hyperactivity-impulsivity that are associated with impairments in multiple functional domains9. We thereby capitalize on the idea that task-induced connectivity patterns build on the baseline functional connectivity architecture as indexed by resting-state MRI analyses12. Using the resting-state architecture as baseline allows assessment of the regional specifics and magnitude of task-induced connectivity modulations across task paradigms9. Specifically, we use task potency as a metric to index the strength of task-induced connectivity modulations in terms of their difference from a resting-state baseline. This approach facilitates the comparison between task paradigms and permits to disentangle modulations that are shared across multiple cognitive functions , resembling a cognitive core15, from those that are task-specific (i.e. Psingle). For example, a comparison of working memory, response inhibition and reward tasks could reveal that participants with ADHD show alterations in the same network of inhibition-related brain regions in each task. This would provide support for theories that claim a prominent role for poor response inhibition in ADHD3. Alternatively, theories suggesting inefficient management of resources in participants with ADHD would be supported by observing, for example, a pattern of modulations that is highly specific to each task, while an overall cognitive core in support of task-general processes remains under-modulated17. Both theories are not incompatible: an alteration in one function network may induce coping strategies involving other functional networks. In combination, this may manifest as inefficiency across multiple task-generic and task-specific systems. In light of these possibilities we hypothesized that, in ADHD, the brain’s functional core interacts differently with more specialized network modulations, which could reflect inefficient use of the brain’s resources in ADHD.Here, we offer such a perspective by applying a novel approach that integrates findings of cognitive tasks across multiple cognitive domains to assess the role of task-dependent connectivity modulationsN = 266) and describe functional connectivity patterns across three cognitive domains . Since unaffected siblings share on average 50% of genetic variation with their ADHD probands, the addition of unaffected siblings allowed assessing the impact of familial vulnerability—addressing the hypothesis that unaffected siblings show a task connectivity modulation profile that is an intermediate phenotype between that of diagnosed siblings and control participants20.To test our hypothesis, we applied our task-potency framework to a large cohort of participants with ADHD, their unaffected siblings, and healthy controls , and at least one of the following task fMRI scans: a spatial working memory task (WM), a monetary-incentive-delay reward task (REWARD) and/or a stop signal response inhibition task (STOP) , and typically developing controls (unrelated to any participant) from the NeuroIMAGE sample9. In brief, for each participant and each pre-processed RS, WM, REWARD and STOP fMRI acquisition (see eMethod for pre-processing procedures) we defined functional connectivity matrices using 179 regions from a hierarchical whole-brain atlas22 . To isolate connectivity changes induced by task modulation  from changes in the brain’s baseline architecture (RS), we standardized each individual-level pair-wise correlation obtained during task acquisition by subtracting the corresponding pair-wise correlation value calculated for the RS scan of that participant. This effectively allows comparing each connection in the task connectivity matrices in terms of its magnitude of deviation from that participant’s resting baseline9. We refer to this deviation as ‘task potency’, which is considered as an absolute value and disregards complex interpretation of down- or up-modulation.Our task potency approach is described in detail elsewheresee Fig. . We calc9 of mixture modelling thresholding25. We used the most conservative limit across groups  in order to compare the same level of information and selected potency values exceeding this limit as being sensitive to task modulation.For each task, we created group-level task potency matrices by averaging the individual-level potency matrices across all participants in each diagnostic group. Within these group-level matrices we selected those connections that were sensitive to task modulation, by thresholding each group-level task potency matrix. Negative and positive thresholds were defined using a two-tailed versionPsingle, to connections that were modulated by more than one but not all tasks as Pmix, and to connections that were modulated regardless of task as Pall. We verified that the relative percentage of participants that performed multiple acquisitions was equivalent between groups to avoid a possible bias related to participant by task interactions in edge selection  versus those connections that were also modulated by one or both of the other groups (shared connections).To account for the heterogeneity in the population, we used a bootstrapping procedure to statistically infer group differences against an appropriate null distribution. Defining empirical null distributions specifically for each diagnostic group allows controlling for sensitivity-differences due to relative group sizes across tasks and across diagnosis. We sub-selected 80% of the study population, computed the different metrics of interest, i.e. true values (percentage of connections per label and amplitude of modulation), randomly relabelled the participants keeping group size equal and computed the same values for a random expectation. We perform this sub-selection and procedure 10,000 times in order to build a null distribution. We tested for significant differences between the average observed values across 10,000 sub-selections and the obtained null distribution (for further details see Supplementary Methods).Pall, Pmix, Psingle connections), and in the percentage of shared versus unique connections for each of the connection labels. To further assess whether differences in percentage of selected connections were associated with different amounts of modulation, we compare their average amplitude of modulation and tested values against the corresponding diagnostic group specific null distribution. P values were assessed for significance using FDR correction across tests per group at q < 0.05. Replication of values in light of possible confounder effects  are presented in Fig. Using this procedure, we tested differences in the percentage of connections modulated across tasks  versus the sharedness (across groups) of each connection. By comparing both rates, we can estimate which connections are uniquely and reproducibly selected in one group only, potentially representing idiosyncratic strategies to solve a task.Finally, we assessed the stability versus the heterogeneity of task connection types in the different groups. To this end we used the 10,000 extracted values from the 80% sample bootstrapping procedure and computed each connection’s selection rate at the group level across bootstraps and its associated shared selection rate between two groups. We computed these rates for sensitive, P < 0.005) compared with the group-specific null distribution , while the percentage of edges modulated by controls and siblings was not significantly different from random expectation .Starting from the set of connections that yielded significant connectivity modulations across all participants, we compared the diagnostic groups in terms of the sensitivity and specificity of their connectome to task modulations. Figure Pall or Psingle , and lower compared to both controls (17.8%) and siblings (16.3%). Within the Pall connections, ADHD seemed to mostly modulate connections that controls and siblings also modulated .Illustrating the task-specific nature of the sensitive connections, the bottom part of Fig. Pall connections, the ADHD group exhibited a significantly higher percentage of Psingle connections compared with the null distribution . In turn, participants with ADHD over-modulated those Psingle connections, suggesting a task-tailored potential compensatory mechanism. In comparison, unaffected siblings of ADHD participants displayed an intermediate phenotype with values in between those observed for controls and ADHD.We used a novel framework to provide an overarching perspective on the neurobiology of ADHD by inferring the nature of connectivity modulations in ADHD under the demands of working memory, reward processing, and response inhibition tasks. Our framework reveals that participants with ADHD activate significantly fewer connections than expected to perform each task. Furthermore, they modulated significantly fewer task-generic connections that share resources across tasks, and instead, relied significantly more on unique sets of connections  larger use of Psingle connections and some differences in the choice of Pall connections compared with controls. In terms of localization of Pall connections (see Fig. 41. Our results support the literature by suggesting that unaffected siblings are potentially still able to recruit more task-general efficient connections.Unaffected siblings of individuals with ADHD share on average 50% of their genetic make-up with the ADHD probands, and accordingly, are hypothesized to share part of the ADHD endophenotype, i.e. biological deficits underpinning the ADHD phenotypesee Fig. , they cosee Fig. . PreviouPsingle connections can be used to investigate such compensatory mechanisms at the level of individual participants. Psingle connections are highly variable across participants, which is also described in previous work on task potency9. For instance, using longitudinal designs and models of compensatory strategies41, we can focus on those connections that are subject-specific and highly reproducible at the individual level to investigate a progressive specialization of individual compensatory mechanisms. In contrast to the high variability of Psingle connections, the absence of some Pall connections was highly reproducible across ADHD and siblings compared with controls (Fig. Pall connections may have potential as a biomarker for ADHD. This would encourage further, out-of-sample investigations of our new task potency approach.The results shown in Fig. ols Fig. . As suchPall connections and the associated differences between ADHD and controls are shown in Fig. 33. These results are coherent with meta-analysis showing hypoconnectivity in fronto-parieto-cingulo-striatal circuit26. As shown in Fig. Pall connections, interestingly involving striatal regions known to be involved in reward processing. Note that these results do not contradict typical findings of aberrant brain activity in reward-related regions in participants with ADHD5 as we showed that participants with ADHD used these connections with greater inconsistency and decreased modulation compared with controls. Knowing that ADHD participants make less efficient use of common pathways to govern multiple cognitive functions, will inform next studies aimed at understanding task response variability in ADHD.Brain areas involved in the 7. As the task modulation builds upon the baseline architecture, after identifying differences between groups during task processing, both levels of baseline and modulation need to be integrated. This will also allow an understanding of the dependency between baseline and task effect and whether task connectivity differences can be predicted on the basis of baseline alterations42. The choice of task in this study is aimed to target cognitive functions altered in ADHD and follow up studies need to extend this finding to other cognitive domains to frame the Pall edges toward specific localized circuit or show a generalization of this effect. In addition, longitudinal studies need to assess age-related trajectories of these findings, as we know that task potency changes with age43. Performance on these tasks also changes with further maturation and changes in symptoms severity that can occur in ADHD with age. The current study does not address a possible developmental delay explanation44, as it would require a longitudinal database to be able to disentangle the complex interaction between symptom severity, age and performance in relation to differences in connectivity.Another important follow up is to integrate knowledge on resting state differencesPall and more Psingle connectivity modulations when compared with controls or unaffected siblings. Our work represents an important step towards new integrative theories explaining how multiple neural alterations interact and result into multiple cognitive impairments in ADHD. Future studies should explore whether the results hold under other tasks paradigms.In conclusion, we examined connectivity modulations across three tasks and demonstrated that individuals with ADHD showed less supplemental material