By 2040, deaths from chronic viral hepatitis worldwide are projected to exceed those from human immunodeficiency virus infection, tuberculosis and malaria combined. The burden of this disease is predominantly carried by low-resource countries in Africa and Asia. In resource-rich countries, the epidemiological spread of viral hepatitis is partially driven by migrant movements from areas of high endemicity. In the last decade, Member States of the European Union and the European Economic Area have experienced an unprecedented influx of migrants, which has resulted in the polarization of political views about migration. In addition, the coronavirus disease 2019 pandemic has worsened the economic and health conditions of migrants and contributed to hostility to ensuring their health rights. Moreover, the implementation of hostile laws in some host nations has increased the vulnerability of marginalized migrant subgroups, such as asylum seekers and undocumented individuals. These developments have complicated the historical challenge of identifying high-risk migrant groups for screening and treatment. However, if European countries can apply the simplified assessment tools and diagnostic tests for viral hepatitis that have been used for decentralized screening and monitoring in resource-poor countries, the uptake of care by migrants could be dramatically increased. Given the global calls for the elimination of viral hepatitis, European nations should recognize the importance of treating this vulnerable migrant population. Political and health strategies need to be adapted to meet this challenge and help eliminate viral hepatitis globally. In 2016, the World Health Organization (WHO) called for an ambitious strategy to eliminate viral hepatitis globally – the initial targets were a 90% reduction in the number of new infections by 2030 compared to 2015 and a reduction of 65% in the number of deaths.,These initiatives and recent research have tended to focus on identifying effective diagnostic and therapeutic modalities and on improving local cascades of care.Migrants’ health and access to care have also been affected by the recent polarization of political views, which has resulted in many European countries becoming resistant to providing health care for migrant subgroups.,Globally only a few countries are on track to achieve viral hepatitis elimination goals and, without greater inclusion of vulnerable migrants in their health-care systems, many European countries are unlikely to achieve these goals within the next decade.Here we discuss barriers to providing viral hepatitis care for vulnerable migrants and present suggestions for improvement.,The global migrant crisis in 2015 saw an unprecedented influx of first-time asylum seekers and undocumented migrants into the European Economic Area. Of the 4.3 million people who immigrated into European Union (EU) Member States during 2015, 1.3 million were first-time asylum seekers from conflict zones around Afghanistan, Iraq and the Syrian Arab Republic who applied for international protection.,,Undoubtedly this was a critical moment when political views on the issue of migration divided European Member States, local communities and even families, ultimately contributing to the increased popularity of several far-right political parties. These polarized views were reflected, in part, by changes in health policy in several EU Member States following the migrant crisis. For example, five EU Member States  and Bosnia and Herzegovina (i) required health-care workers to report undocumented migrants attending clinical facilities to the immigration office; (ii) did not provide state-funded treatment for communicable diseases; and (iii) restricted access to services other than emergency care.Lancet Commission observed that migrants arriving in Europe often had better health than people who remained in their countries of origin – this was termed the “healthy migrant concept”.Lancet Commission’s study recognized that some subgroups from specific geographical locations, who may have been vulnerable to infectious liver diseases in their home nations or during their journeys, were more likely to have or be at risk of contracting these diseases.,In response to concerns that migrants were major contributors of communicable diseases within host nations, a recent Contrary to their sworn oaths of beneficence and non-maleficence, health-care workers have been unwillingly recruited into a political battle in which the provision of basic care has become conditional. The issue of viral hepatitis sits at a critical juncture in this complex situation because migrant flows tend to come from regions where viral hepatitis is highly endemic and because the nature of the care cascade for the condition touches on aspects of screening, linkage to various services, patient retention and the provision of long-term treatment. Any strategy that can effectively address viral hepatitis could be applicable to other communicable diseases associated with migration.The novel, affordable treatments provide a unique opportunity to eliminate viral hepatitis globally. Furthermore, the replacement of liver biopsies by non-invasive tests has enabled a major obstacle in the care cascade to be overcome. However, the impact of these developments has been tempered by the need for an effective public health strategy to ensure adequate disease prevention, monitoring and treatment coverage. Predominantly, tertiary care centres manage patients with HCV infection. These centres have strict treatment eligibility criteria, which may involve the diagnosis of cirrhosis, measurement of the HCV viral load, detection of the HCV genotype and the need to abstain from drugs and alcohol before accessing HCV therapy. However, there is no evidence for the necessity of drug and alcohol abstention, which has been a major obstacle to receiving care.The complexity of current models of care presents barriers to all migrant groups. In addition, particular subgroups, such as undocumented migrants and asylum seekers, face further obstacles: they may be excluded from the general health-care system because host nations have implemented hostile laws or because sociocultural factors deter them from accessing care. Despite these hurdles, there are several reasons why these subgroups must be targeted if viral hepatitis is to be eliminated in the European Economic Area or, indeed, globally.First, the burden of viral hepatitis, especially of HBV-associated hepatitis, in the European Economic Area is driven by migrant movements from highly endemic nations. In 2016, it was estimated that 53% of 49 million migrants born outside of the European Economic Area came from nations where the endemicity of HBV infection was either intermediate (i.e. above 2%) or high (i.e. above 8%).,,,Second, chronic viral hepatitis follows a prolonged asymptomatic course during which infected individuals are unaware of their infection, until they reach an advanced disease stage. About 40–80% of people with chronic hepatitis virus infections worldwide are unaware of their infective status.Finally, challenges exist even after screening for viral hepatitis has been performed. Linking individuals to care and, thereafter, maintaining contact with them for long-term monitoring or therapy have presented difficulties in various settings worldwide. The development of simpler techniques for measuring liver fibrosis (e.g. transient elastography) has made the task easier.In summary, certain migrant subgroups are: (i) more at risk of viral hepatitis because of their previous exposure to specific risk factors; (ii) more at risk of their condition being undiagnosed than they would have been in their native country or compared with the general population in their host nation; and (iii) more at risk of failing to be retained within the health-care system because of complex social factors and growing hostility within the system. Consequently, these subgroups must be specifically targeted to ensure they receive effective treatment and, moreover, to enable viral hepatitis to be eliminated within Europe. In many ways, migrant communities in host nations exist in a milieu similar to that in their native countries. Reaching these communities may, therefore, depend on using robust, local measures that have been tried and tested before in similar settings internationally.,Although the management of viral hepatitis varies enormously around the world, reaching marginalized migrant communities may be possible by adopting innovations that focus on decentralizing and expanding care and treatment. There are many ways in which this can be achieved. In low-resource settings, for example, where access to laboratory facilities can be difficult, guidelines on the management of infectious diseases have helped simplify care for HIV-infected patients by advocating innovative, low-technology, analytical techniques, such as dried blood spot testing to assess viral loads.,Task-shifting, that is, specialized jobs are performed by less-specialized workers, has been crucial for scaling up services and improving treatment coverage for other infectious diseases, such as tuberculosis and HIV infection. This approach counters the disadvantages  of clinical services being concentrated in tertiary care centres. Clearly, the need for task-shifting is greatest in low-resource regions where there are critical shortages of most health-care workers – some countries have 100 times fewer doctors per capita than EU nations. For HBV and HCV infections, the availability of effective treatments with few side-effects considerably increases the possibility of task-shifting. A meta-analysis showed that task-shifting increased uptake of HBV testing fourfold in migrant communities when it was performed by culturally appropriate health-care workers.,–Jointly with task-shifting, targeting specific communities and adopting a proactive approach to screening have helped identify migrants with viral hepatitis and subsequently link them to care.,The transition to community-based diagnosis and monitoring requires tools that can be accessed by decentralized facilities, while ensuring they remain diagnostically valid. Simple laboratory scores have been developed and used in low-resource regions as alternative tools for assessing liver disease severity and treatment eligibility: they include (i) the aspartate aminotransferase-to-platelet ratio index (APRI); (ii) the FIB-4 score; and (iii) the TREAT-B score, which is based on hepatitis B virus e antigen serostatus and the alanine aminotransferase level.If these assessment tools and diagnostic tests can be applied in European countries to provide a decentralized screening and monitoring system for displaced and marginalized communities, including high-risk migrant subgroups, the uptake and continuity of care for these individuals could be dramatically increased. Once this step has been achieved, the emphasis should be on providing neglected communities with the normal standard of care, without prejudice or political bias. This implementation must be rigorously monitored.In this interconnected world, complex sociopolitical factors hinder the reach of migrant communities in Europe who require treatment for viral hepatitis. Moreover, development of strategies addressing the consequences of rapid global movements of people into the European Economic Area from regions where viral hepatitis is highly endemic is essential. Relative to the impact of the current COVID-19 pandemic, viral hepatitis ultimately forms a small part of the overall discourse on population health. Nevertheless, if international efforts and global policies can be directed towards addressing this substantial public health concern, the result may be better understanding of how to improve health care as a whole for marginalized groups in Europe. Overall, the changes required begin and end with our perception of, and our attitude towards, people in these groups. Health-care workers have a particular responsibility when dealing with the most vulnerable in society and could challenge laws that state otherwise.


Low- and middle-income countries (LMICs) face disproportionately high mortality rates, yet the causes of death in LMICs are not robustly understood, limiting the effectiveness of interventions to reduce mortality. Minimally invasive tissue sampling (MITS) is a standardized postmortem examination method that holds promise for use in LMICs, where other approaches for determining cause of death are too costly or unacceptable. This study documents the costs associated with implementing the MITS procedure in LMICs from the healthcare provider perspective and aims to inform resource allocation decisions by public health decisionmakers.We surveyed 4 sites in LMICs across Sub-Saharan Africa and South Asia with experience conducting MITS. Using a bottom-up costing approach, we collected direct costs of resources  to conduct MITS and the pre-implementation costs required to initiate MITS.Initial investments range widely yet represent a substantial cost to implement MITS and are determined by the existing infrastructure and needs of a site. The costs to conduct a single case range between $609 and $1028 per case and are driven by labor, sample testing, and MITS supplies costs.Variation in each site’s use of staff roles and testing protocols suggests sites conducting MITS may adapt use of resources based on available expertise, equipment, and surveillance objectives. This study is a first step toward necessary examinations of cost-effectiveness, which may provide insight into cost optimization and economic justification for the expansion of MITS. The vast majority of global childhood deaths are disproportionately concentrated in low- and middle-income countries (LMICs)  and crafEvidence documenting the validity , 11, 12,To date, no comprehensive estimates of the costs to implement MITS in LMIC settings have been published, leading to calls for more evidence . The majSeveral studies reference costs to conduct MITS in LMIC settings but do not conduct a comprehensive accounting of the costs. An examination of barriers to MITS implementation in Mozambique noted that complex microbiology platforms, the need for specialized labor, and the high cost of specific supplies like biopsy needles all present challenges to MITS expansion . Feroz eIn this work, we aim to characterize the costs and cost drivers associated with conducting MITS in LMIC settings, by examining 4 sites where MITS has been implemented at pilot scale. As a result, this case study approach speaks to the potential costs of MITS, rather than generalizable findings. We capture costs from the perspective of the health care provider or health system in order to provide national health ministries and agencies with information on what MITS implementation may cost in order to inform efficient resource allocation decisions.This study adopts a micro-costing approach to capturing costs associated with MITS from the healthcare provider perspective. “Bottom-up” micro-costing estimates the unit resources  at the detailed activity level, and aggregates them to form summary totals . This apWe adopt the healthcare provider perspective because in LMICs, as in many countries, mortality surveillance activities are primarily conducted by healthcare providers. Providers’ activities are directed and funded by national heath ministries, public agencies, or other nonprofit entities interested in efficient resource allocation. For this reason, we acknowledge but do not include costs from the family member or societal perspectives, which often entails unexpected and often large costs to the family of the deceased.In consultation with subject matter experts, we identified key activities required to conduct MITS in a facility  and in the community, and designed a survey to capture the associated unit-level direct costs that were incurred . Direct We surveyed 4 sites conducting MITS at university hospitals in LMICs across Sub-Saharan Africa and South Asia. All sites were grantees of the MITS Surveillance Alliance Secretariat that received incentive grants to expand the use of MITS. Participating sites had experience conducting MITS (>10 MITS cases completed at the time of data collection) and represented varied case populations. We collected data in LMICs between February and August of 2020, before and during the early stages of the coronavirus disease 2019 (COVID-19) pandemic. At each site, the principal investigator and an employee knowledgeable of the site’s finances responded to the computer-based survey. The teams provided additional details and clarifications by follow-up teleconference and email. Each team reviewed and approved the final cost estimates. At sites where data were collected after COVID’s onset, we asked them to report the costs incurred prior to the pandemic, which most readily had affected the price of personal protective equipment.Respondents provided resource information for activities associated with preparing the site to conduct MITS and conducting the MITS procedure, based on actual costs incurred rather than budgets or projected expenses . This inTo capture labor expenses, respondents entered all staff roles that typically contributed to the preparation or performance of MITS. For every staff role, teams indicated the number of persons performing the role, their compensation, and their typical hours worked per week at their site. Respondents provided the number of hours that staff members typically spent on a given activity for an average MITS case. Respondents recorded these hours separately from time spent on administrative tasks related to MITS, like grant management, quarterly reporting, and equipment maintenance, for which we do not account in this study. We compared role-specific wage estimates given by sites to Serje et al’s estimates of health worker earnings as multiples of gross domestic product (GDP) per capita in low- and lower-middle income countries , findingWe developed an Excel-based cost model to aggregate each sites’ activity-specific resource cost estimates and categorized them into 1-time, initial costs  required to be in place for a MITS program to begin and recurring per-case and annual costs to conduct MITS at each site. Costs provided in local currencies were converted to US dollars. We provide a financial accounting of these costs —that is, we did not adjust for depreciation and presented the purchase cost for these items that had been purchased recently or through the MITS Surveillance Alliance grant. We excluded costs to perform additional research activities.The majority of cost categories required simple aggregation. Staff compensation varied by site, including with regular salaries or hourly wages, fixed incentive payments per-case, and per-diem payments for specific activities. We calculated activity-specific labor costs by activity, based on each site’s reported compensation schedule. Where staff were paid hourly wages, we multiplied their wage by the number of hours they spent on an activity, whereas for salaried staff we estimated an hourly wage where appropriate. Start-up costs included some nonconsumable expenditures like equipment purchases and facility renovations that were not used exclusively for MITS. In these cases, we adjusted these costs by the utilization rate of the MITS program. Site-specific estimates were reviewed with respondents before finalizing results.Labor costs constitute roughly 25% of MITS costs per case. Although regional wage differences explain some differences across sites, sites reported a high degree of heterogeneity in staff roles participating in an average MITS procedure . All sitCosts to conduct sample analysis, or testing, make up a significant portion (18%) of the per case cost and are determined by the type and number of tests conducted per case, which varied by the site-specific protocols . All sitMITS kits, which are provided to sites with the needed supplies to conduct the MITS procedure, comprise the largest portion (48%) of the per case MITS cost. This cost includes a standard battery of supplies ($225), a back-up kit sent with each batch of 10 kits ($29 per kit), and assembly and shipping costs to send kits from the United States ($142 labor and shipping). Sites received these kits directly from the MITS Surveillance Alliance program.The 1-time costs required to establish a MITS program, divided into start-up activity and capital costs, far outweigh the cost to complete a single case and differ substantially by site. The wide variation in capital costs by site is driven primarily by the preexisting infrastructure available. Renovation expenses, where necessary, drove capital costs, as some sites adapted existing lab and office space to accommodate MITS. Other capital expenses included equipment and machinery. Site 1 was least well-resourced initially and required a costly autopsy table and embedding machine, whereas site 4 had fewer needs, investing only in a microscope.Start-up costs varied notably by site and reflect each site’s investment in specific pre-implementation activities. For instance, the volume of staff trained in each site differed, which reflects a diversity of available staff roles and/or those selected to receive MITS training. Guidelines shared by the MITS Surveillance Alliance did not stipulate a specific number of hours to complete pre-implementation activities yet did provide standards for their completion.The purpose of this cost analysis was to understand the cost implications of MITS implementation in a range of different LMIC contexts, providing a range of transparent cost estimates that reflect the resources needed for expanding MITS in new locations. These cost estimates are an initial milestone in demonstrating the comprehensive costs of conducting MITS incurred by a provider. Our findings identify important cost drivers and opportunities for reducing cost.The lack of standardization across sites in terms of labor and testing—key drivers of incremental costs—suggests that staff roles and testing protocols may be varied without sacrificing the accuracy of cause of death findings. This variation points to the potential flexibility around labor and testing to reduce per case costs and adapt MITS protocols around resource availability or surveillance goals. First, the notable variation in staff roles applied by each site has the potential to both give greater flexibility to sites that do not have a large number of specialty clinicians available and points to opportunities to reduce labor costs. For instance, with a core team of pathologists for supervision, other staff roles could perform some tasks in place of pathologists. Forthcoming research related to “task-shifting” across roles to conduct MITS explores this in-depth and is an important area of future study.Second, the varied type and number of tests conducted across the 4 sites suggests that improved insight into causes of death from MITS can still be achieved through nonuniform sample testing protocols. Each site’s protocols reflect site-specific surveillance objectives, testing budgets, and the equipment and expertise available. In our study, sites used standard testing procedures across the sites, but no site used more advanced tests, like TAC PCR and traditional PCR tests. This may be due to the limited availability of testing equipment, such as PCR machines, or because more tests may not imply improved cause of death information. Due to the initial investment in infrastructure to conduct tests using molecular techniques like PCR, their inclusion would likely increase the cost to perform MITS, despite the decreasing costs of PCR, in part due to widespread PCR testing for COVID-19. A growing body of evidence suggests that incremental testing may increase data associated with the presence of pathogens yet may not necessarily increase the precision and accuracy of cause of death determination . Ritter As MITS is conducted at relatively few locations globally, expanding its use gives opportunity to reduce per case costs by procuring supplies locally. Although the costs associated with MITS kits are substantial—between 35% and 65% of per case costs—they reflect MITS implementation at pilot-scale and could be reduced in the future by local procurement. The contents of the kits are not highly specialized, suggesting that they may not be limited by supply chains in LMICs.We find that incremental (per case) costs to conduct MITS were relatively minor, once the initial investments to perform MITS were made. This is positive, in that low incremental costs may avoid the challenges of disinvestment over time, as many public health funders may be wary of securing substantial long-term resources to sustain highly costly programs.The initial investments varied by site due to the differing needs and existing infrastructure for capital goods to conduct MITS and highlights the importance of evaluating the infrastructure in-place when considering costs to conduct MITS at a new site. Where necessary, renovation of office and lab space comprised a large portion of these costs, as substantial space is needed to conduct MITS. However, no capital expense was required universally by site, suggesting that potential locations may have the majority of necessary items on hand. Activities to prepare for MITS implementation, including training, reflected each site’s ability to devote resources and/or priority given to these activities.This study represents a small number of sites, and although each site’s costs and context vary, they illustrate the diversity in approaches to implement MITS. As such, obtaining precisely comparable information from each site was difficult. Although we conduct limited cross-site comparisons in this analysis, the individual differences of the economies and healthcare environments limit the utility of these comparisons. Comparing specific resources or staffing roles may be most telling for contextualizing these findings in a new location. We also do not discuss the effectiveness of the MITS program. In the future, it will be important to weigh the body of evidence for MITS to assess the value in replacing existing routine surveillance systems.Our study did not include all possible costs of MITS implementation, such as family counseling, socio-anthropological support , and clinical data abstraction. Importantly, our study also did not include program administration costs , which are a fundamental component of MITS program operational expenses. We were, however, careful to exclude them from our estimates. Administrative costs are notoriously challenging to include and interpret, and often favor a “top-down” costing methodology to account for administrative burden, which is often less precisely tracked and can be under-reported in “bottom-up” costing . FurtherFinally, all activities and costs represented in this study are defined by external grant funding and the technology available to each site. Thus, these results may not represent the activities that would be conducted in the absence of a budget constraint or at expanded scale, but do represent illustrative examples of MITS implementation in LMIC settings where these constraints are common. For instance, sites implementing MITS beyond a pilot-scale may increase community sensitization work in order to improve MITS’ acceptability and overall success. The scale and funding of the costs represented could be both higher and lower than the costs of self-funded MITS implementation.This study is a first step toward additional necessary examinations of cost considerations, including cost-comparison and cost-effectiveness studies, which further provide needed economic justification for the expansion of MITS for improving cause of death data.


REPLY: We would like to thank van de Stadt and colleagues for their positive and encouraging comments regarding our study recently published in The Journal of Nuclear Medicine . There is evidence that brain tumors or metastases display a focally disrupted BBTB, which could lead to enhanced brain tumor delivery of anticancer agents as compared with healthy brain tissue. In clinical practice, BBTB disruption is assessed based on MRI measurements with gadolinium-containing contrast agents. However, these contrast agents possess physicochemical properties distinct from molecularly targeted anticancer agents and are not likely to be transported by ABCB1 and ABCG2. Therefore, brain delivery of MRI contrast agents may not be representative of brain delivery of molecularly targeted anticancer agents. Consequently, PET using the radiolabeled anticancer agents themselves in tumor patients is clearly a powerful approach, as illustrated by some recent studies ,5.In summary, the use of PET with molecularly targeted anticancer agents, which has been pioneered by the group at the VUmc in Amsterdam, has great potential to address and refine pharmacokinetic issues related to the design of brain-targeted anticancer drugs.


Coronavirus pandemic has affected all medical institutions, and dental schools are no exception . DentistAcademic dental schools are known to be demanding and highly competitive learning environments. COVID-19 pandemic highlighted the differences between medical and dental institutions in the practical and clinical educational activities. Academic dental institutions are known as competitive and demanding learning communities, where students study preclinical and clinical dental courses designed to equip them with the essential knowledge and skills for licensure and clinical practice . ClinicaAs we are starting the third wave of COVID-19 pandemic, dental academic institutions in Egypt must learn from previous experiences and take the professional responsibility to share information. We hope that dental schools will be able to maintain adequate training in a safe clinical setting. Regarding clinical training, it is important to answer many questions that may help in understanding our future actions like are dental schools considered among high-risk workplaces or they should be treated like other educational in-campus settings? What will be the basic infection control standards? Are there any plans regarding the COVID-19 vaccination? Are there expected modifications on the future designs and infrastructure of educational dental settings including waiting areas, and spacing of dental units, and the central air ventilation? Although hybrid learning will probably be a keystone of future dental education, it is crucial to study extensively the possible pedagogical effects of the sudden change in dental educational strategies caused by the COVID-19 crisis. Finally, as an educator, I wish to go back soon to normal interactive dental education, but I believe that dental education might change significantly in the near future.


Neurocognitive function (NCF) deficits are common in patients with brain metastases, occurring in up to 90% of cases. NCF deficits may be caused by tumor-related factors and/or treatment for the metastasis, including surgery, radiation therapy, chemotherapy, and immunotherapy. In recent years, strategies to prevent negative impact of treatments and ameliorate cognitive deficits for patients with brain tumors have gained momentum. In this review, we report on research that has established the efficacy of preventative and rehabilitative therapies for NCF deficits in patients with brain metastases. Surgical strategies include the use of laser interstitial thermal therapy and intraoperative mapping. Radiotherapy approaches include focal treatments such as stereotactic radiosurgery and tailored approaches such as hippocampal avoidant whole-brain radiotherapy (WBRT). Pharmacologic options include use of the neuroprotectant memantine to reduce cognitive decline induced by WBRT and incorporation of medications traditionally used for attention and memory problems. Integration of neuropsychology into the care of patients with brain metastases helps characterize cognitive patterns, educate patients and families regarding their management, and guide rehabilitative therapies. These and other strategies will become even more important for long-term survivors of brain metastases as treatment options improve. The most common metastases to the brain are from lung cancer, breast cancer, melanoma, and renal cell cancer.2 These patients face difficult treatments, including neurosurgery, radiation therapy, and additional rounds of systemic therapy.As treatments for systemic cancer have improved, the proportion of patients experiencing metastatic disease to the central nervous system continues to increase. The development of CNS metastases, estimated to occur in up to 30% of patients with systemic cancer,3 can be caused the tumor itself and by the treatments applied to both systemic and CNS disease. Numerous aspects of NCF, including memory, processing speed, and executive function, are disrupted in patients with brain metastases prior to any CNS directed treatment,4–6 and successful treatment of brain metastases can lead to improvement in neurologic symptoms attributable to the disease itself and forestall the inevitable decline associated with tumor progression. Treatment of brain metastases requires a multimodal approach and may include radiation therapy, surgery, chemotherapy, and newer techniques such as immunotherapy or laser interstitial thermal ablative therapy. While the goal of these therapies is to improve progression-free and overall survival, they can also cause brain injury leading to neurocognitive dysfunction. Understanding the mechanisms of how these therapies can damage healthy brain tissue is vital for educating our patients and their caregivers about these impairments and developing strategies to mitigate or prevent the injury.Loss of neurocognitive function (NCF), which has been shown to occur in as many as 90% of patients with brain metastases,7), take care of a family, manage a household, and even care for oneself.8–10 In a study examining the relationship between NCF, QOL, and activities of daily living (ADLs),11 NCF deficits were strongly associated with problems with ADLs and QOL in patients with brain metastases who were treated with whole-brain radiation therapy (WBRT). After treatment, declines in NCF preceded and predicted ADL and QOL deterioration, which occurred in a substantial percentage of brain metastasis patients . The NCF deficits experienced by patients with brain metastases constitute a threat to the individual in a way that is distinct from the other symptoms of cancer, striking at their identity and sense of independence,12 undermining their sense of productivity and meaning in life, financial security, and potentially limiting their access to care if health insurance coverage through an employer is lost. Many patients with brain metastases have a poor prognosis, whereas others are benefiting from new treatments that are extending progression-free and overall survival. In either case, it is incumbent upon treatment providers to optimize the patient’s QOL, providing the best possible treatment of disease and minimizing the deleterious impact of those treatments on NCF.Declines in NCF are associated with a significant impact on the individual’s quality of life (QOL), including losing the ability to perform a job, safely operate a motor vehicle  to target solitary and oligo-metastatic disease, which reduces the risk of NCF impairment without compromising progression and survival endpoints.13 Key mediators of neurotoxic inflammation include tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8). This inflammation can occur during or immediately after RT, leading to the acute cognitive side effects and the chronic white matter loss seen in patients exposed to RT with late loss of microglia.14 Radiologically, these phenomena can manifest as acute inflammation during radiation therapy and widespread leukoencephalopathy years after radiation therapy is completed.15 In concert with increased neuroinflammation, radiation disrupts the normal functioning of neural progenitor cells, particularly in the region of the hippocampus, a neural structure of the temporal lobe that is critical for learning and memory.16RT fundamentally leads to DNA damage via the generation of reactive oxygen species. In addition to the production of DNA double-strand breaks and single-strand breaks in tumor cells, all components of healthy cells are damaged by radiation. The primary mechanisms of this damage are likely activation of inflammation of the neural tissue along with activation of microglia, which represent CNS-derived macrophage-like cells.17 Clinically, these deficits may manifest as “forgetfulness,” with patients requiring lists and reminders, and often dependence on caregivers to compensate for these deficits and their impact on ADLs. As a considerable minority of patients are long-term survivors following WBRT, strong data are lacking regarding more long-term neurocognitive outcomes, however, clinically the usual course is of plateauing or slow decline in memory, with more dependence on caregivers for ADLs.Historically, neurocognitive decline has been described as occurring at different stages during and after treatment with WBRT: acute changes that occur within days of initiation of treatment, subacute changes evolving in the weeks after treatment through the first few months after completion, and chronic/progressive changes, which generally begin to appear about 6 months after treatment and lead to inexorable deterioration. More recently, randomized trial data from patients undergoing treatment for brain metastases with or without the use of WBRT have demonstrated that many patients show declines in memory and executive functioning by 4 months after treatment.17 The pivotal multicenter phase III clinical trial4 included 213 individuals with 1–3 brain metastases. Subjects were randomized to either receive SRS or WBRT plus SRS. Patients who received only SRS had better NCF outcomes both at an early time point  and for long-term survivors . Although the WBRT group had lower rates of brain metastasis recurrence (distant control), there was no significant difference in overall survival .Recognizing the importance of RT in the management of brain metastases, diverse strategies have been explored to mitigate the cognitive morbidity of treatment. The goal of these strategies has been to achieve equivalent disease control while reducing cognitive side effects by using strategies such as focal irradiation, avoidance of critical neural structures, and neuroprotectant therapies. Promising results from early studies suggested that individuals with brain metastases treated with focal RT such as SRS  have bet18 as well as the role of the hippocampus in neuronal regeneration and plasticity, an additional neuroprotective strategy has been to modify treatment to selectively avoid the hippocampal region  receptor antagonist that reduces harmful excessive stimulation of NMDA receptors has been shown to reduce neurotoxicity of radiation therapy.23 Brown et al.24 found those who received memantine during WBRT showed longer time to cognitive decline than those who did not receive memantine. The memantine group also had stronger performance on measures of executive functioning 16 weeks later and better processing speed and delayed recognition at 24 weeks.24 More recently, the combined use of memantine and HA-WBRT25 was demonstrated to further reduce the frequency of NCF decline in a large phase III clinical trial.The use of pharmacological agents to protect against radiation-induced cerebral injury has been an additional strategy to reduce neurocognitive morbidity. Memantine, an 5 Concomitant neurologic sequelae, including edema, seizures, and headaches, can also contribute to NCF dysfunction. Furthermore, the medications to treat these complications can have adverse effects on neuronal function. While neurological injury leading to NCF decline is possible during neurosurgical procedures26 due to trauma to the local healthy brain tissue, to the extent that resection reduces mass effect, edema, or disruption of CSF flow, surgery can lead to improved performance status maintained over a longer period of time.27 Similar to radiation-induced damage, studies have found that after brain surgery, rats and mice demonstrate increased neural tissue inflammation with resulting induction of TNF-α and IL-8.29 Additionally, brain-derived neurotrophic factor (BDNF) is reduced, with a consequent decrease in hippocampal neurogenesis. These changes are accompanied by NCF impairments in murine behavioral tests.30 Xin et al. have shown that by inhibiting proinflammatory signaling pathways, in particular nitrous oxide (NO) pathways, one can rescue postoperative cognitive dysfunction in mice and rats.31 Developing methods to limit neuroinflammation after surgery has the opportunity to provide protection against and mitigation of neurocognitive dysfunction.The impact of a metastatic tumor on the CNS relates to the overall burden of disease in the brain, most accurately quantified as the overall volume of brain disease.32 Technological advances, such as intraoperative mapping of cognitive function during awake craniotomy, provide an opportunity for the monitoring of NCF during the procedure. Although awake craniotomy is more commonly employed in the resection of primary brain tumors, a recent review found that awake craniotomy for brain metastases was a viable option to reduce cognitive morbidity.33 The review showed that surgery in/near eloquent cortex leads to increased risk of postoperative neurocognitive deficits as compared with surgeries farther from eloquent regions; however, 73% of patients undergoing awake craniotomy were not found to have a decline on a brief bedside neurologic exam conducted by the surgeon. Of those who had a decline in the acute postoperative period, 96% showed subsequent improvement and recovery.Neurocognitive risk of surgery is greatest when brain metastases arise near eloquent areas, particularly speech/language and memory-related areas in the dominant hemisphere.34 Heating above 50°C leads to cell death. LITT is now becoming more widely used in patients with brain metastases. Similar to the injury induced by traditional neurosurgical procedures, anatomical location and proximity to eloquent areas or areas involved in cognition are vital to understand how LITT could impact NCF. One can glean information on long-term cognitive data by looking at the use of LITT in epilepsy patients. Small nonrandomized studies point out that memory decline can occur in patients with dominant medial temporal lobe epilepsy who underwent LITT involving the hippocampus,35 but these patients were spared the language declines often seen in patients who undergo standard resective surgery for this condition. In patients with brain metastases, where the target of LITT is not functional neural tissue, the hope is that this approach could lead to reduced neurocognitive morbidity in difficult to reach areas of the brain compared with resective surgery. A study of 39 patients, 20 of whom had brain metastases (19 more had radiation necrosis) by Ahluwalia et al. demonstrated no reduction in neurocognitive performance.36 The role of LITT in patients with brain metastases continues to be explored. Balancing the risks and benefits of surgery along with application of these new techniques in eloquent areas will continue to be the aim for neurosurgical procedures in brain metastases patients.Laser interstitial thermal therapy (LITT) involves neurosurgical stereotactic placement of a laser probe that kills tumor tissue with heat.37 and adverse neurocognitive outcomes have been found in survivors of treatment for childhood leukemia.38 Capecitabine effects on neurocognition have been reported but are generally mild,39 while paclitaxel effects have also been of concern, given anti-microtubule mechanism and known association with peripheral neuropathy and acute encephalopathy.40 An extensive literature demonstrates the neurotoxicity of these and many other traditional chemotherapy agents , which may manifest as acute or subacute neurocognitive syndromes or more subtle cognitive deficits that are longer lasting . Multiple mechanisms have been proposed for these effects, including inflammatory mechanisms, direct cellular toxicity, myelin damage, and loss of hippocampal neurogenesis.42 Patients with brain metastases receiving high-dose or intrathecal methotrexate are further at risk for the development of methotrexate-induced leukoencephalopathy, demonstrated by NCF impairment in the setting of T2/FLAIR hyperintensities on magnetic resonance imaging, particularly when combined with radiation therapy.43 In preclinical rat and murine models, exposure to methotrexate (with or without 5-fluoruracil) leads to NCF dysfunction, which was attributable to loss of neurons in the hippocampus and frontal lobes.44 More recent work demonstrates that damage to pathways involved in oligodendrocyte integrity and adaptive myelination via BDNF signaling is responsible for cognitive impairment in mice exposed to methotrexate.45In concert with other therapeutics, use of systemic agents in patients with brain metastases is expanding rapidly with the list of FDA approved targeted therapies enlarging. The trend for chemotherapy in treatment of brain metastases lies prominently with targeted agents. Regardless, there is continued use of agents such as methotrexate, capecitabine, and paclitaxel, all of which have been found to affect NCF. For example, neurotoxicity of methotrexate therapy has been well demonstrated in adult CNS lymphoma patients46 Theoretically, these effects reflect the ubiquitous expression of estrogen and androgen receptors in key areas of the brain involved in cognition, such as prefrontal cortices and the hippocampus.47 A recent review of this literature46 concluded that there is evidence of cognitive impairment in patients with breast or prostate cancer after treatment with hormonal therapies. A longitudinal prospective study followed women with breast cancer who either were or were not treated with hormonal therapies found no difference in cognitive symptoms or performance up to 6 years posttreatment,48 though this analysis was limited to group comparisons and may have missed possible individual differences in response to therapy. In men undergoing androgen deprivation therapy (ADT) for prostate cancer, a longitudinal prospective study showed no differences in cognitive performance over a 3-year period between patients who were treated with ADT, patients who did not need such treatment, and healthy controls.49 However, a similar study found increased risk for cognitive decline in patients treated with ADT and identified a genetic risk factor that appeared to markedly increase risk in a subset of patients.50 Thus, it appears that there are cognitive risks associated with hormone therapies and additional research is needed to identify the relevant risk factors and longer term outcomes.While hormone-based therapies are not classic chemotherapy, these treatments are commonly used in the treatment of breast and prostate cancer and may contribute to cognitive impairment.51 The long-term implication for NCF function is best determined when one considers concomitant use of radiation therapy. McGinnis et al. developed preclinical models in mice exposed to immunotherapy, particularly immune-checkpoint inhibitors (ICIs) with and without concomitant radiation therapy.52 All mice that received the combination of immunotherapy and radiation developed cognitive impairment and notably, this cognitive impairment was in the setting of tumor control. Mechanistically, microglia appeared to be activated by immunotherapy, with or without concomitant radiation.Immunotherapy has revolutionized the treatment of patients with melanoma and other cancers that regularly send metastases to the central nervous system; however, encephalitis due to autoimmune induced inflammation in the brain can lead to both acute and chronic neurologic impairments.3 Identification of individuals at greatest risk of NCF morbidity is an important aspect of treatment decision making. Advanced age and a higher degree of pretreatment leukoencephalopathy are both associated with a greater risk of cognitive dysfunction in patients with brain metastases who received WBRT,54 suggesting that health of the underlying brain tissue contributes to NCF risk in these patients. Researchers have initiated studies into the genetic factors associated with the risk of NCF dysfunction in patients with primary brain tumors and those who received treatment for CNS and non-CNS cancers. Variations on a theme that implicates apolipoprotein E (APOE), a gene related to the risk of Alzheimer’s disease, are present in the literature pertaining to cancer related cognitive impairment (CRCI) in breast cancer patients.55 Unfortunately, the literature is at odds as to whether APOE genotype is meaningful in predicting risk of CRCI. Preliminary work suggests that those with a high-risk APOE genotype experience greater cognitive decline when they undergo WBRT than do those with a lower risk genotype.56 Correa et al.58 showed that specific SNPs in catechol-O-methyl transferase (COMT), BDNF, and dystrobrevin-binding protein 1 (DTNBP1) genes can be associated with dysfunction in a myriad of cognitive domains. How these translate specifically to patients with brain metastases remains to be determined.Despite efforts to reduce the cognitive risks of therapy detailed above, many patients with brain metastases will experience cognitive symptoms during the course of their disease, requiring a comprehensive clinical strategy for management.59) and the Montreal Cognitive Assessment (MoCA60) have only modest ability to detect symptoms in brain metastasis patients.62 Thus, a careful clinical inquiry regarding these symptoms is an important first step in assessing cognitive function during clinic visits. Self-report surveys can be used to inquire about subjective NCF changes in the context of QOL assessment with scales such as the Functional Assessment for Cancer Therapy-Brain (FACT-Br63) and the European Organization for Research and Treatment of Cancer Quality of Life Scale (EORTC-QLQ-C3064), including newly developed metrics that are moderately correlated with cognitive complaints.65 Neuropsychological (NP) evaluation is the most sensitive method of identifying cognitive dysfunction in patients with cancer, and specifically sensitive tests has been recommended by the International Cognition and Cancer Task Force (ICCTF).66 Neuropsychological evaluations have been flexibly integrated in the neuro-oncology clinic, including in metastatic brain tumor boards.67 These evaluations, often abbreviated to minimize burden on the patient,68 are sensitive to NCF changes and can detect progression of brain metastases prior to MRI.69 Integrating NP evaluations in the care of patients with brain metastases provides an understanding of NCF and related symptoms, recommendations for treatment, and guidance to the patient and family and is recommended in the most recent guidelines issued for CNS cancers by the National Comprehensive Cancer Network .70Monitoring NCF in the neuro-oncology clinic is difficult for clinicians because brief screening measures, such as the Mini-Mental State Examination  was a randomized placebo-controlled trial that included 53 patients (25%) with brain metastases72 and measured cognitive effects at 12 and 24 weeks of treatment. In the 74% of patients who completed follow-up visits (% of brain metastases not reported), there were subtle indications of a treatment effect on one measure of recognition memory.An early study of the memory enhancer donepezil in a mixed group of patients with brain tumors had only one brain metastasis patient at baseline, who failed to complete the follow-up assessments,73 randomized placebo-controlled trials failed to replicate the findings, including the only study to include patient with brain metastases,74 suggesting that expectancy effects may play a significant role in the experience of patients prescribed these medications. It should be noted that this study evaluated fatigue, rather than cognitive function, as the primary endpoint.Attention-enhancing medications, such as methylphenidate and modafinil, have been evaluated in mixed groups of brain tumor patients, though these studies too have largely excluded patients with brain metastases. Although early studies of this approach suggested some benefits,75 and goal management training for executive function problems.76 Other approaches, such as combining the training of compensatory strategies with “cognitive exercise” activities have shown at least partial benefit in randomized controlled studies.77 Studies using remote methods  to deliver rehabilitation have also shown promise,78 including a method for cognitive stimulation leading to improved NCF test performance.79 To date, only two cognitive rehabilitation studies have included patients with brain metastases, both of which used variations of cognitive exercise training80 These small studies reported positive impacts of cognitive training but are limited to some extent by lack of a control group80 and small sample size.79 While these studies are opening doors for new methods, it has yet to be demonstrated that improvements on NCF tests or computerized exercises translate to benefits in the real world. Additional approaches to improve cognition in patients with brain tumors have included physical rehabilitation,81 which demonstrated a positive effect on MMSE scores in patients with brain tumors (including metastatic) in the weeks after surgery. There are hopes that other strategies such as exercise82 and herbal strategies83 may prove to be helpful, though the prevailing view in the field is that findings are too preliminary to form the basis of recommendations at this time.84Cognitive rehabilitation is the use of therapeutic strategies to minimize the impact of NCF deficits on everyday functioning and/or improve cognitive function, which may include education in compensatory strategies as well as massed practice of cognitive exercises intended to provide neurocognitive stimulation. The majority of these studies have evaluated cognitive rehabilitation in patients with primary brain tumors and have suggested some benefit to those patients who receive training in specific cognitive strategies, such as the use of mnemonic strategies for memory problems86 enable in vivo validation and testing of candidate genes and drugs, as well as histopathology and imaging studies.87–89 Perhaps most importantly for preclinical research, elegant work to refine behavioral and neurocognitive testing in laboratory animals, often aided by complementary research from fields such as Alzheimer’s disease, developed assays such as the novel object recognition test and Morris water maze and correlative functional tests such as roto-rod to presage neurocognitive endpoints in humans.91The continued need for radiation therapy in the treatment of brain tumors and the increasing prevalence of brain metastases drives research into improving NCF for patients receiving brain radiotherapy. Model systems spanning the in vitro and in vivo spaces, including conventional and 3D culture systems, lend themselves to exploratory studies for neuroprotectors, while small animal radiation platforms and adaptations of clinical radiation therapy equipment24 Combinations of memantine with AMPA receptor inhibitors are now planned for patients with primary brain tumors receiving RT92 and could soon extend to patients with brain metastases. Recently, development of manganese porphyrin compounds that alter the redox biology of mitochondria has generated interest as possible dual tumor radiosensitizers and normal tissue radioprotectors.93 Preclinical findings of preserved tumor control and neuroprotection with enhanced cognitive function following irradiation in mice spawned clinical trials in several cancer types, including brain metastases87 (NCT03608020). The role of GSK-3beta inhibition as a general neuroprotection strategy that prevents radiation necrosis88 is also exciting, and some clinical trial data exist for tideglusib in Alzheimer’s disease patients.95 Many other exciting data for novel drugs that target pathways such as hedgehog signaling96 and modulation of the complement cascade97 also show potential to improve neurocognitive outcomes in brain metastasis patients receiving radiotherapy.Numerous clinical trials are currently studying a wide range of approaches to improve cognitive outcomes in brain metastasis patients see . On the 25 and further technical advances in the administration of SRS simultaneously to multiple target lesions promises to expand this technique for more patients with a high burden of brain metastases (NCT02886572). Upcoming trials will further differentiate the advantages of these approaches, and results are anxiously awaited (NCT03550391). While these techniques utilize the most advanced radiation therapy technologies that are in current clinical use, a newer technique has recently emerged that seeks to maximally exploit the fundamental differences in radiation biology responses that distinguish tumor from normal tissue. “Flash” radiation therapy employs ultra-high-dose rate radiation delivery (40–100 Gy/s) to harness a theoretical difference in normal tissue responses to radiation that has implications for radiotherapy to multiple areas of the body, including the brain.99 Although this approach is not yet available for widespread use, the first clinical tests of this technology and development of clinical instruments look promising.101Another strategy toward improving neurocognitive outcomes in patients receiving brain-directed RT leverages advanced radiation therapy techniques. Some of these techniques are in use currently and involve precise control over radiation dose deposition such that anatomical regions of the brain are spared damage. As noted above, HA-WBRT and SRS have shown neurocognitive benefit16 Neural stem cell transplantation is theoretically possible, and with current stem cell technologies one can contemplate autotransplant of a patient’s own induced neural stem cells. Preclinical studies indicate that this approach could be beneficial103 and might be warranted in the increasingly plausible case that long-term cancer control in brain metastasis patients is attainable.Lastly, biotechnology strategies that push the limits of current science focus on radiation-induced loss of neural stem cells in the hippocampus.Neurocognitive sequelae are an unfortunate reality for most patients with brain metastases, which can be caused by the metastatic tumors, treatment for the systemic disease, and treatment directed at the brain. Numerous advances over the past two decades, including neurosurgical techniques, focal delivery of radiotherapy, and neuroprotectant strategies, have reduced the negative impact on the brain. Integration of NP assessment in the routine care of patients with brain metastases allows for monitoring of cognitive outcomes and tailoring of treatment. Rehabilitative therapies and pharmacologic treatment of cognition are useful options for patients. As therapeutic options for cancer and brain metastases continue to improve, the focus on neurocognitive outcomes of the long-term survivors will become even more important.The following case example illustrates the multiple opportunities to integrate many of the techniques we have described in the clinical care of a patient with brain metastases to optimize cognitive outcome. The patient, a 67-year-old Caucasian man, initially developed a mass on the left upper back and underwent resection, with pathology confirming malignant melanoma. He had been treated with combination immunotherapy (ipilimumab + nivolumab) for approximately 3 months when he developed altered mental status. Brain imaging at the time was unrevealing, and the patient was suspected to be experiencing ICI-related encephalitis. He underwent treatment with intravenous immunoglobulin and neuro-rehabilitation during a 2-month hospitalization and ultimately recovered, though experienced a slightly reduced level of cognitive functioning compared with his normal baseline. ICI therapy was discontinued. Unfortunately, about 6 months later, surveillance brain imaging showed a subcentimeter enhancing lesion in the left lateral temporal lobe, which was felt to represent melanoma metastasis . Based oAt the time of SRS, the patient reported persistent changes in his cognitive function compared with his usual baseline. Neuropsychological evaluation was requested at that time. During the interview, the patient reported difficulties with concentration and the ability to hold information in mind while multitasking . He also described deficits in recent memory, such as forgetting conversations or things that he had agreed to do. He and his wife felt that these problems had been present since he recovered from ICI encephalitis and had not changed significantly since the new development of brain metastasis or SRS treatment of that lesion. The NP evaluation demonstrated that the patient was a man of above average premorbid ability who was experiencing relative deficits in aspects of attention, including lower than expected encoding of new information into memory , likely Over the ensuing year, the patient was treated with imatinib and systemic melanoma was well controlled. Unfortunately, approximately 1 year after SRS, the left temporal lesion showed increased size and contrast enhancement as well as intratumoral hemorrhage and increased surrounding edema in the left temporal lobe . It was In consultation with the patient, the decision was made to resect the lesion. A functional magnetic resonance imaging study was conducted to identify foci of critical language activity in the left hemisphere and identified a site of putative receptive language function ~1 cm from the lesion boundary. Diffusion tensor imaging was conducted during the same MRI session to identify critical fiber tracts and demonstrated the location of the arcuate fasciculus passing within 7 mm of the mass. These imaging studies were fused with the structural imaging in the surgical navigation software. Intraoperative mapping of language function demonstrated an area 1 cm superior to the lesion in which stimulation produced deficits in language comprehension. The cortex overlying the lesion was tested with stimulation mapping, and no changes were elicited in comprehension, naming, or reading. A gross total resection was achieved with pathology demonstrating recurrent melanoma. The patient remained awake throughout the procedure and demonstrated no gross deficits postoperatively.The patient was seen for a repeat NP evaluation ~1 month after surgery. At that point, he reported good recovery of function and had returned to his part time professional role as a technical advisor to a biotechnology firm. He and his wife reported improved memory compared with the preoperative time point but acknowledged increased fatigue and reduced cognitive endurance since surgery. The evaluation demonstrated a significant improvement in memory as compared with the preoperative assessment, returning to the level of performance seen 14 months prior . There w


Liquid biopsy with circulating tumor DNA (ctDNA) profiling by next-generation sequencing holds great promise to revolutionize clinical oncology. It relies on the basis that ctDNA represents the real-time status of the tumor genome which contains information of genetic alterations. Compared to tissue biopsy, liquid biopsy possesses great advantages such as a less demanding procedure, minimal invasion, ease of frequent sampling, and less sampling bias. Next-generation sequencing (NGS) methods have come to a point that both the cost and performance are suitable for clinical diagnosis. Thus, profiling ctDNA by NGS technologies is becoming more and more popular since it can be applied in the whole process of cancer diagnosis and management. Further developments of liquid biopsy ctDNA testing will be beneficial for cancer patients, paving the way for precision medicine. In conclusion, profiling ctDNA with NGS for cancer diagnosis is both biologically sound and technically convenient. It is now well known that a tumor can shed cancer cells, extracellular vesicles, proteins and nucleic acids into the peripheral blood . These tctDNA is shed into the peripheral blood by tumor cells in cancer patients. However, the biological mechanisms of how ctDNA enters and presents in the blood circulation have not yet been fully elucidated . GeneralApart from tumor-specific molecular characteristics, studies have also found that the levels of ctDNA in advanced stage cancers such as breast, colorectal, pancreatic, and gastroesophageal cancers are higher than in the early stage of diseases . HoweverBecause the ctDNA represents only a small part of the cfDNA, it is important to utilize the tumor-specific alterations to distinguish themselves from their wild-type counterparts. Currently, most methods using ctDNA as cancer biomarkers focus on mutation de tection . MutatioNGS for ctDNA analysis can be targeted or untargeted as well . TargeteFor the sequencing step, the cost, accuracy, and speed are all key factors that need to be taken into consideration for ctDNA profiling. It is particularly true for diagnosis purposes. Under this circumstance, there are currently several platforms more suitable for ctDNA sequencing, including Illumina’s sequencing-by-synthesis (SBS), Thermo Fisher Scientific’s Ion Torrent, and the nanopore sequencing from Oxford Nanopore Technologies ,22. IlluThe key for precisely identifying ctDNA from a large amount of background cfDNA is to correctly detect the alterations of ctDNA compared to normal cfDNA and single nucleotide mutation is the most commonly detected alteration . TechnicBecause ctDNA is mainly distinguished from normal cfDNA by tumor-specific mutations, it is thus critical to identify a true mutation for sensitive ctDNA detection. Plus, the low levels of ctDNA presented in the bloodstream also warrant highly sensitive and specific methods that can detect and quantify mutant alleles in cfDNA. This can be achieved both by experimental designs and bioinformatics algorithms. Some of these strategies are presented below to elucidate the general principles.Tagged-amplicon deep sequencing (TAm-Seq) introduces a two-step amplification strategy to first amplify regions of interest by multiplex PCR using specifically designed primers at low concentrations to avoid inter-primer interactions, followed by using a microfluidic system in a second amplification step to amplify individual target regions using the amplicons from the first step . SequencUnique molecular identifiers (UMIs) are often used to barcode individual templates so that the amplicons from different templates can be identified . UMIs arA combination of optimized library preparation and bioinformatics algorithm is often used to achieve better performance of NGS assays for low-abundance sequence aberration in ctDNA analysis. Target enrichment of DNA fragments containing recurrent mutations combined with deep sequencing can also improve the sensitivity of mutation detection. CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) is such an NGS assay that combines a target-selection library preparation approach with specialized bioinformatics workflow . It geneAlthough early diagnosis of cancer is the key to reduce morbidity and mortality of cancer, it is still challenging due to technical limitations, especially for the broad diagnosis of different types of cancer. The postulation of using ctDNA for early cancer diagnosis is that tumors will release fragmented nucleic acids into the circulating bloodstream before they can be visualized by other diagnosis approaches or cause perceptible symptoms. Therefore, early diagnosis of cancer by analyzing ctDNA alterations requires both sensitivity and specificity. If the method can also determine the origin of the ctDNA, it is likely to be an ideal method. However, this demands that the ctDNA detection technique identifies tumor-specific mutations from background cfDNA and their normal alteration in a minute amount of DNA fragments.Many cancer patients are asymptomatic at the early stage of diseases. For example, pancreatic cancer is a very aggressive disease, but most patients are diagnosed at the late stage with advanced metastasis. Additionally, due to the lack of therapeutic options for advanced disease, pancreatic cancer patients have extremely poor 5-year survival. However, math ematical modeling of the mutation acquisition rate suggests that the development of pancre atic cancer is extremely slow in that an 11.7-year period from acquisition of the initiating mutation to full transformation in a pancreatic cell, and another 6.8 years are needed to develop the first metastatic subclone. Thus, early diagnosis is the key to improve the prog nosis of pancreatic cancer patients . MutatioCancerSEEK is a blood test that aims for early diagnosis of different types of cancer ,39. It iLiquid biopsy for ctDNA testing seems to give promising biomarkers for asymptomatic early cancer diagnosis. However, theoretical calculations based on reported tumor measures show that when the fraction of tumor DNA falls below 0.01% of the total cfDNA, drawing 10 mL of blood  for ctDNA testing will likely to contain less than one cancer genome . In suchGenetic mutations found in the genomic DNA that can be treated with available anticancer therapeutics or the target of developing new drugs are called targetable mutations or actionable mutations. Targetable mutations are clinically relevant because it is the ultimate goal to guide treatments for the detection of mutations in ctDNA . Many ofTumor-specific mutations in the coding region of genes can produce mutant proteins which are not present in normal cells. These mutant proteins, called neoantigens, can activate the immune system to attack the cancer cells . NeoantiTo implement precision methods for cancer therapeutic strategies, there is an increasing interest in disease prognosis using genomic alterations . ctDNA fThe efficacy of neoadjuvant chemotherapy (NAC) can be evaluated by ctDNA profiling. Preliminary results of a study showed that combined profiling of ctDNA by NGS with functional tumor volume (FTV) by magnetic resonance imaging (MRI) improves the prediction of metastatic recurrence and death of patients treated with NAC . They foTumor evolution can also be accessed by NGS ctDNA profiling. A study in non-small cell lung cancer (NSCLC) patients demonstrated that more mutations were detected in targetable genes by ctDNA than tissue biopsy samples . AnotherMinimal residual disease or measurable residual disease (MRD) is a term used for describing the presence of a small number of tumor cells in the body after treatment. MRD is responsible for the relapse of cancer ,90. ThusBecause new generation sequencing is high-throughput, sensitive, specific and relatively cost-efficient nowadays, mutation profiling of ctDNA by NGS technologies is now showing its advantages over other techniques. It has already been demonstrated by many studies and commercial services that, NGS-based ctDNA profiling can potentially help to detect cancer early, effectively identify actionable mutations, as well as to help with the prognosis of cancer patient outcomes. Thus, NGS ctDNA profiling is already transforming molecular oncology to precision medicine. With technical advancements alongside price reduction, it can be foreseen that the NGS-based liquid biopsy for ctDNA profiling will accelerate its pace to be widely adopted in routine clinical diagnosis.


Therapeutic proteins such as monoclonal antibodies and their derivatives, fusions proteins, hormone analogs and enzymes for replacement therapy are an ever-growing mainstay in our pharmacopoeia. While a growing number of these medications are developed for and used in younger and younger pediatric patients, knowledge gaps in the basic understanding of the molecular and physiologic processes governing the disposition of these compounds in the human body and their modulation by age and childhood development are a hindrance to the effective and timely development and clinical use of these compounds, especially in very young pediatric patient populations. This is particularly the case for the widespread lack of information on the ontogeny and age-associated expression and function of receptor systems that are involved in the molecular processes driving the pharmacokinetics of these compounds. This article briefly highlights three receptor systems as examples, the neonatal Fc receptor, the asialoglycoprotein receptor, and the mannose receptor. It furthermore provides suggestions on how these gaps should be addressed and prioritized to provide the field of pediatric clinical pharmacology the urgently needed tools for a more effective development and clinical utilization of this important class of drugs with rapidly evolving importance as cornerstone in pediatric pharmacotherapy. Over the past 25 years, therapeutic proteins such as monoclonal antibodies (mAbs) and their derivatives, fusions proteins, hormone analogs and enzymes for replacement therapy have gained major roles in the armamentarium to treat numerous conditions and diseases . More reWhile pediatric dosing may be affected by differences in pharmacokinetic as well as pharmacodynamic processes but also differences in disease etiology and progression, particular interest has often been directed towards pharmacokinetic differences. This is based on the notion that full and partial extrapolation approaches of efficacy from adults to children frequently rely on exposure-matching where dosing regimens of the drug in question in different pediatric populations are selected in such a way that they achieve drug exposures in the pediatric patients “similar” to those having shown to be efficacious and safe in adults . This apThe pharmacokinetic processes of distribution and elimination of therapeutic proteins are governed by combinations of physicochemical, physiologic and receptor-mediated processes and have been reviewed in detail elsewhere . In brieElimination processes can broadly be distinguished into unspecific proteolytic degradation that can either be receptor-mediated or non-receptor-mediated . Non-recPediatric extrapolation efforts to establish dosing regimens for therapeutic proteins are hampered by a lack of a comprehensive understanding of the differences in drug distribution and elimination mechanisms between children and adults, particularly young pediatric patients such as full term and premature neonates and infants, i.e., in the range younger than 1 year of age. While many disposition processes based on physicochemical and physiologic processes are reasonably well understood, those related to receptor-mediated processes remain in many instances unclear or elusive. In more general terms, size-related differences between children and adults have relatively well been characterized, while knowledge on pediatric maturation-related differences remains spotty.The distribution processes of most therapeutic proteins, as described in the previous section, are largely driven by conserved physicochemical processes together with physiologic differences between adults and different pediatric age groups and can thus usually be well predicted for pediatric populations. Therefore, allometric scaling approaches accounting for body size differences between children and adults usually characterize the distribution of therapeutic proteins well. Only for very young pediatric patients such as newborns and infants, further differences may need to be considered. These include the well-known higher total and extracellular tissue water content, larger capillary beds and thus capillary surface area per tissue volume, and higher perfusion rates . All theSimilar to distribution volumes, clearance values for non-receptor-mediated proteolytic degradation processes of therapeutic proteins in children can also relatively well be derived from adult values based on allometric scaling with allometric exponents of 0.75 or 0.85 accounting solely for body size-based differences between children and adults . Only foFor receptor-mediated elimination processes, however, the available knowledgebase on age- and maturation-related differences between children and adults is very scarce. For FcRn, for example, data have been limited to rodent studies. While messenger RNA (mRNA) expression of the α-chain of FcRn in rats suggested an age-associated increase , more reAn example for a promiscuous membrane receptor facilitating the uptake of therapeutic proteins for subsequent lysosomal degradation is the asialoglycoprotein receptor (ASGPR) . It is eSimilar to ASGPR, the mannose receptor is a highly effective endocytic receptor that is expressed on selected populations of macrophages and dendritic cells, and that recognizes glycoproteins with mannosylated glycan chains . High-maFor target-mediated drug disposition processes, data are even more scarce than for those elimination processes related to less specific receptor systems such as ASGPR or the mannose receptor. One might expect that each target has its own specific ontogeny with age- and maturation-associated differences in target receptor abundance, turnover and internalization kinetics . This beThe selection of safe and efficacious dosing regimens for drug development and applied pharmacotherapy of therapeutic proteins in pediatric patients is severely hampered by substantial knowledge gaps on the ontogeny and age-associated expression and function of receptor systems that are involved in the molecular processes driving the pharmacokinetics of these compounds. This is particularly relevant for newborns and infants where differences in therapeutic protein pharmacokinetics cannot be fully explained by size differences between children and adults and where additional maturation processes need to be considered. This article briefly highlighted three receptor systems as examples, FcRn, ASGPR and the mannose receptor, but numerous others may be involved in the disposition process of specific therapeutic proteins as well. Priorities for filling these knowledge gaps should be initially directed towards those receptor systems that are more broadly relevant to the largest number of therapeutic proteins, for example, FcRn for all mAbs and mAb derivatives with intact FcRn binding site.Population pharmacokinetic modeling (PopPK) and physiological pharmacokinetic modeling (PBPK) have been widely used in support of pediatric extrapolation exercises based on exposure-matching approaches for traditional small molecule drugs . While PThere have recently been elegant attempts to impute the lack of age-associated function of receptor systems such as FcRn through PBPK modeling frameworks using known PK data of endogenous and therapeutic proteins . While t


Oxytocin (OT) is a neuropeptide produced by hypothalamic neurons and is known to modulate social behavior among other functions. Several experiments have shown that OT modulates neuronal activity in many brain areas, including sensory cortices. OT neurons thus project axons to various cortical and subcortical structures and activate neuronal subpopulations to increase the signal-to-noise ratio, and in turn, increases the saliency of social stimuli. Less is known about the origin of inputs to OT neurons, but recent studies show that cells projecting to OT neurons are often located in regions where the OT receptor (OTR) is expressed. Thus, we propose the existence of reciprocal connectivity between OT neurons and extrahypothalamic OTR neurons to tune OT neuron activity depending on the behavioral context. Furthermore, the latest studies have shown that OTR-expressing neurons located in social brain regions also project to other social brain regions containing OTR-expressing neurons. We hypothesize that OTR-expressing neurons across the brain constitute a common network coordinated by OT. Mouse pvia whisker trimming or dark rearing after birth leads to reduced synaptic transmission in somatosensory and visual cortices, respectively, as well as to abolished OT synthesis, release, and overall OT neuron activity , nucleus accumbens (NAcc), amygdala (Amy), and lateral habenula (LHb; Peris et al., When summarizing all known connections between areas containing OTR neurons (as we have endeavored to do in Several recent technological developments render possible the exploration of these questions. For instance, using pseudotype rabies infecting cells in a cre dependent manner combined with transgenic OTR-Cre animals could bring evidence that OTR neurons in one region are innervated by OTR neurons from another region. Another useful tool is the generation of non-toxic pseudotype rabies (Ciabatti et al., In this review, we proposed two novel features of OT signaling in the brain. First, we showed that OT neurons received reciprocal input from the OT-sensitive structures they are innervating. Although the functional significance of such potential feedback projections is not clear, it is tempting to postulate that OTR neurons of distant brain regions dynamically tune “positive” vs. “negative” balance of OT neurons to initiate, accelerate, or slow down behavioral responses. Secondly, we hypothesized that OTR neurons in spatially distant brain regions are communicating with each other to further control social behaviors elicited by an initial OT release. An important note is that all the studies reviewed here have been performed in rodents, and thus whether the proposed circuits might also exist in other mammals is unknown. Finally, we would like to point out that OT is the most studied neuropeptide at the moment, but that the mechanisms proposed here can be expanded towards other less renown neuropeptides, which are also important neuromodulators of social behaviors (Lefevre et al., All authors discussed and elaborated the ideas, wrote the first draft and corrected it until the final version was obtained. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.