More than a small adult brain: Lessons from chemotherapy-induced cognitive impairment for modelling paediatric brain disorders.

Childhood is recognised as a period of immense physical and emotional development, and this, in part, is driven by underlying neurophysiological transformations. These neurodevelopmental processes are unique to the paediatric brain and are facilitated by augmented rates of neuroplasticity and expanded neural stem cell populations within neurogenic niches. However, given the immaturity of the developing central nervous system, innate protective mechanisms such as neuroimmune and antioxidant responses are functionally naïve which results in periods of heightened sensitivity to neurotoxic insult. This is highly relevant in the context of paediatric cancer, and in particular, the neurocognitive symptoms associated with treatment, such as surgery, radio- and chemotherapy. The vulnerability of the developing brain may increase susceptibility to damage and persistent symptomology, aligning with reports of more severe neurocognitive dysfunction in children compared to adults. It is therefore surprising, given this intensified neurocognitive burden, that most of the pre-clinical, mechanistic research focuses exclusively on adult populations and extrapolates findings to paediatric cohorts. Given this dearth of age-specific research, throughout this review we will draw comparisons with neurodevelopmental disorders which share comparable pathways to cancer treatment related side-effects. Furthermore, we will examine the unique nuances of the paediatric brain along with the somatic systems which influence neurological function. In doing so, we will highlight the importance of developing in vitro and in vivo paediatric disease models to produce age-specific discovery and clinically translatable research.

Methodological and ethical challenges in the use of focused ultrasound for blood-brain barrier disruption in neuro-oncology.

BACKGROUND: Focused ultrasound (FUS) shows promise for enhancing drug delivery to the brain by temporarily opening the blood-brain barrier (BBB), and it is increasingly used in the clinical setting to treat brain tumours. It remains however unclear whether FUS is being introduced in an ethically and methodologically sound manner. The IDEAL-D framework for the introduction of surgical innovations and the SYRCLE and ROBINS-I tools for assessing the risk of bias in animal studies and non-randomized trials, respectively, provide a comprehensive evaluation for this. OBJECTIVES AND METHODS: A comprehensive literature review on FUS in neuro-oncology was conducted. Subsequently, the included studies were evaluated using the IDEAL-D framework, SYRCLE, and ROBINS-I tools. RESULTS: In total, 19 published studies and 12 registered trials were identified. FUS demonstrated successful BBB disruption, increased drug delivery, and improved survival rates. However, the SYRCLE analysis revealed a high risk of bias in animal studies, while the ROBINS-I analysis found that most human studies had a high risk of bias due to a lack of blinding and heterogeneous samples. Of the 15 pre-clinical stage 0 studies, only six had formal ethical approval, and only five followed animal care policies. Both stage 1 studies and stage 1/2a studies failed to provide information on patient data confidentiality. Overall, no animal or human study reached the IDEAL-D stage endpoint. CONCLUSION: FUS holds promise for enhancing drug delivery to the brain, but its development and implementation must adhere to rigorous safety standards using the established ethical and methodological frameworks. The complementary use of IDEAL-D, SYRCLE, and ROBINS-I tools indicates a high risk of bias and ethical limitations in both animal and human studies, highlighting the need for further improvements in study design for a safe implementation of FUS in neuro-oncology.

A phase I/II study of bevacizumab, irinotecan and erlotinib in children with progressive diffuse intrinsic pontine glioma.

INTRODUCTION: This study investigates the safety, tolerability, and preliminary efficacy of combined treatment with VEGF inhibitor bevacizumab, topoisomerase I inhibitor irinotecan, and EGFR inhibitor erlotinib in children with progressive diffuse intrinsic pontine glioma (DIPG). METHODS: Biweekly bevacizumab (10 mg/kg) and irinotecan (125 mg/m2) were combined with daily erlotinib. Two cohorts received increasing doses of erlotinib (65 and 85 mg/m2) following a 3 + 3 dose-escalation schedule, until disease progression with a maximum of one year. Dose-limiting toxicities (DLT) were monitored biweekly. Secondary progression free survival (sPFS) and overall survival (OS) were determined based on clinical and radiological response measurements. Quality of life (QoL) during treatment was also assessed. RESULTS: Between November 2011 and March 2018, nine patients with disease progression after initial radiotherapy were enrolled. Median PFS at start of the study was 7.3 months (range 3.5-10.0). In the first dose cohort, one patient experienced a DLT (grade III acute diarrhea), resulting in enrollment of three additional patients in this cohort. No additional DLTs were observed in consecutive patients receiving up to a maximum dose of 85 mg/m2. Median sPFS was 3.2 months (range 1.0-10.9), and median OS was 13.8 months (range 9.3-33.0). Overall QoL was stable during treatment. CONCLUSIONS: Daily erlotinib is safe and well tolerated in doses up to 85 mg/m2 when combined with biweekly bevacizumab and irinotecan in children with progressive DIPG. Median OS of the study patients was longer than known form literature.

Complementary and alternative medicine in children with diffuse intrinsic pontine glioma-A SIOPE DIPG Network and Registry study.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is a rare and aggressive childhood brainstem malignancy with a 2-year survival rate of <10%. This international survey study aims to evaluate the use of complementary and alternative medicine (CAM) in this patient population. METHODS: Parents and physicians of patients with DIPG were asked to participate in a retrospective online survey regarding CAM use during time of illness. RESULTS: Between January and May 2020, 120 parents and 75 physicians contributed to the online survey. Most physicians estimated that <50% of their patients used CAM, whereas 69% of the parents reported using CAM to treat their child during time of illness. Cannabis was the most frequently used form of CAM, followed by vitamins and minerals, melatonin, curcumin, and boswellic acid. CAM was mainly used with the intention of direct antitumor effect. Other motivations were to treat side effects of chemotherapy or to increase comfort of the child. Children diagnosed from 2016 onwards were more likely to use CAM (χ2  = 6.08, p = .014). No significant difference was found between CAM users and nonusers based on ethnicity (χ2  = 4.18, p = .382) or country of residence (χ2  = 9.37, p = .154). Almost 50% of the physicians do not frequently ask their patients about possible CAM use. CONCLUSION: This survey demonstrates that worldwide, a considerable number of patients with DIPG use CAM. Physicians should be more aware of potential CAM use and actively discuss the topic. In addition, more research is needed to gain knowledge about possible anticancer effects of CAM and (positive/negative) interactions with conventional therapies.

Correction to: International experience in the development of patient-derived xenograft models of diffuse intrinsic pontine glioma.

There are two errors and one omission in the original article. Author Gottardo's correct name is Nicholas G. Gottardo, author Hulleman's correct affiliation is no. 3 (VUMC, Amsterdam), and the Acknowledgements should include the following sentence: "We would like to thank Dr Angel Montero Carcaboso (Hospital Sant Joan de Deu, Barcelona, Spain) for generously supplying the HSJD-DIPG007 cells."

International experience in the development of patient-derived xenograft models of diffuse intrinsic pontine glioma.

PURPOSE: Diffuse intrinsic pontine glioma is the most aggressive form of high grade glioma in children with no effective therapies. There have been no improvements in survival in part due poor understanding of underlying biology, and lack of representative in vitro and in vivo models. Recently, it has been found feasible to use both biopsy and autopsy tumors to generate cultures and xenograft models. METHODS: To further model development, we evaluated the collective international experience from 8 collaborating centers to develop DIPG pre-clinical models from patient-derived autopsies and biopsies. Univariate and multivariate analysis was performed to determine key factors associated with the success of in vitro and in vivo PDX development. RESULTS: In vitro cultures were successfully established from 57% of samples (84.2% of biopsies and 38.2% of autopsies). Samples transferred in DMEM media were more likely to establish successful culture than those transported in Hibernate A. In vitro cultures were more successful from biopsies (84.2%) compared with autopsies (38.2%) and as monolayer on laminin-coated plates than as neurospheres. Primary cultures successfully established from autopsy samples were more likely to engraft in animal models than cultures established from biopsies (86.7% vs. 47.4%). Collectively, tumor engraftment was more successful when DIPG samples were directly implanted in mice (68%), rather than after culturing (40.7%). CONCLUSION: This multi-center study provides valuable information on the success rate of establishing patient-derived pre-clinical models of DIPG. The results can lead to further optimization of DIPG model development and ultimately assist in the investigation of new therapies for this aggressive pediatric brain tumor.

Diagnostics and treatment of diffuse intrinsic pontine glioma: where do we stand?

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is a rare clinically, neuro-radiologically, and molecularly defined malignancy of the brainstem with a median overall survival of approximately 11 months. Our aim is to evaluate the current tendency for its treatment in Europe in order to develop (inter)national consensus guidelines. METHODS: Healthcare professionals specialized in DIPG were asked to fill in an online survey with questions regarding usual treatment strategies at diagnosis and at disease progression in their countries and/or their centers, respectively. RESULTS: Seventy-four healthcare professionals responded to the survey, of which 87.8% were pediatric oncologists. Only 13.5% of the respondents biopsy all of their patients, 41.9% biopsy their patients infrequently. More than half of the respondents (54.1%) treated their patients with radiotherapy only at diagnosis, whereas 44.6% preferred radiotherapy combined with chemotherapy. When the disease progresses, treatment strategies became even more diverse, and the tendency for no treatment increased from 1.4% at diagnosis to 77.0% after second progression. 36.5% of the healthcare professionals treat children younger than 3 years differently than older children at diagnosis. This percentage decreased, when the disease progresses. Most of the participants (51.4%) included less than 25% of their patients in clinical trials. CONCLUSION: This survey demonstrates a large heterogeneity of treatment regimens, especially at disease progression. We emphasize the need for international consensus guidelines for the treatment of DIPG, possible by more collaborative clinical trials.

High occurrence of sleep problems in survivors of a childhood brain tumor with neurocognitive complaints: The association with psychosocial and behavioral executive functioning.

BACKGROUND: Survivors of childhood brain tumors are prone to sleep and neurocognitive problems. Effective interventions to improve neurocognitive functioning are largely lacking. In general, sleep problems are negatively related to neurocognitive functioning, but this relationship is unclear in survivors of childhood brain tumors. Therefore, the occurrence of sleep problems, potential risk factors, and the relation between sleep and executive functioning were evaluated. PROCEDURE: Baseline data of a randomized controlled trial on the effectiveness of neurofeedback were used. Childhood brain tumor survivors 8-18 years of age with parent-reported neurocognitive complaints ≥2 years after treatment were eligible. Parents completed the Sleep Disturbance Scale for Children. Executive functioning was assessed by parents and teachers (Behavior Rating Inventory of Executive Functioning). Multiple linear regression analyses were used to examine sociodemographic and medical characteristics and emotional difficulties and hyperactivity/inattention (Strength and Difficulties Questionnaire) as potential risk factors for sleep problems, and to assess the association between sleep and executive functioning. RESULTS: Forty-eight percent of survivors (n = 82, 7.0 ± 3.6 years post diagnosis, age 13.8 ± 3.2 years) had sleep problems and scored significantly worse than the norm on the subscales Initiating and Maintaining Sleep, Excessive Somnolence, and the total scale (effect sizes 0.58-0.92). Emotional problems and/or hyperactivity/inattention were independent potential risk factors. Sleep problems were associated with worse parent-reported executive functioning. CONCLUSIONS: Sleep problems occur among half of childhood brain tumor survivors with neurocognitive problems, and are associated with worse executive functioning. Future studies should focus on the development of sleep interventions for this population, to improve sleep as well as executive functioning.

Biological material collection to advance translational research and treatment of children with CNS tumours: position paper from the SIOPE Brain Tumour Group.

Paediatric CNS tumours are the most common cause of childhood cancer-related morbidity and mortality, and improvements in their diagnosis and treatment are needed. New genetic and epigenetic information about paediatric CNS tumours is transforming the field dramatically. For most paediatric CNS tumour entities, subgroups with distinct biological characteristics have been identified, and these characteristics are increasingly used to facilitate accurate diagnoses and therapeutic recommendations. Future treatments will be further tailored to specific molecular subtypes of disease, specific tumour predisposition syndromes, and other biological criteria. Successful biomaterial collection is a key requirement for the application of contemporary methodologies for the validation of candidate prognostic factors, the discovery of new biomarkers, the establishment of appropriate preclinical research models for targeted agents, a quicker clinical implementation of precision medicine, and for other therapeutic uses (eg, for immunotherapies). However, deficits in organisational structures and interdisciplinary cooperation are impeding the collection of high-quality biomaterial from CNS tumours in most centres. Practical, legal, and ethical guidelines for consent, storage, material transfer, biobanking, data sharing, and funding should be established by research consortia and local institutions to allow optimal collection of primary and subsequent tumour tissue, body fluids, and normal tissue. Procedures for the collection and storage of biomaterials and related data should be implemented according to the individual and organisational structures of the local institutions.

Correction to: A suggestion to introduce the diagnosis of "diffuse midline glioma of the pons, H3 K27 wildtype (WHO grade IV)".

The citation of the original publication in PubMed contains an error. The seventh author name is wrongly cited.

Correction to: A phase I/II study of gemcitabine during radiotherapy in children with newly diagnosed diffuse intrinsic pontine glioma.

In Table 2 of the original publication, there were errors in the baseline scores for the PedsQL TM 3.0 Cancer Module questionnaire, so a corrected version of Table 2 is shown in this erratum. In the subcategories of the PedsQL TM 3.0 Cancer Module questionnaire, nausea and fear of procedure did not score significantly lower after treatment compared to baseline. So, based on the corrected data in Table 2, there was no significant decrease in the total score of the cancer questionnaire, and this statement in the previous manuscript was incorrect.

Culture methods of diffuse intrinsic pontine glioma cells determine response to targeted therapies.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive type of brainstem cancer occurring mainly in children, for which there currently is no effective therapy. Current efforts to develop novel therapeutics for this tumor make use of primary cultures of DIPG cells, maintained either as adherent monolayer in serum containing medium, or as neurospheres in serum-free medium. In this manuscript, we demonstrate that the response of DIPG cells to targeted therapies in vitro is mainly determined by the culture conditions. We show that particular culture conditions induce the activation of different receptor tyrosine kinases and signal transduction pathways, as well as major changes in gene expression profiles of DIPG cells in culture. These differences correlate strongly with the observed discrepancies in response to targeted therapies of DIPG cells cultured as either adherent monolayers or neurospheres. With this research, we provide an argument for the concurrent use of both culture conditions to avoid false positive and false negative results due to the chosen method.

A phase I/II study of gemcitabine during radiotherapy in children with newly diagnosed diffuse intrinsic pontine glioma.

The purpose of this phase I/II, open-label, single-arm trial is to investigate the safety, tolerability, maximum tolerated dose and preliminary efficacy of the potential radiosensitizer gemcitabine, administered concomitantly to radiotherapy, in children with newly diagnosed diffuse intrinsic pontine glioma (DIPG). Six doses of weekly gemcitabine were administered intravenously, concomitantly to 6 weeks of hyperfractionated radiotherapy. Successive cohorts received increasing doses of 140, 175 and 200 mg/m2 gemcitabine, respectively, following a 3 + 3 dose-escalation schedule without expansion cohort. Dose-limiting toxicities (DLT) were monitored during treatment period. Clinical response was assessed using predefined case report forms and radiological response was assessed using the modified RANO criteria. Quality of life (QoL) was assessed using PedsQL questionnaires. Between June 2012 and December 2016, nine patients were enrolled. Treatment was well tolerated, and no DLTs were observed up to the maximum dose of 200 mg/m2. All patients experienced reduction of tumor-related symptoms. QoL tended to improve during treatment. PFS and MOS were 4.8 months (95% CI 4.0-5.7) and 8.7 months (95% CI 7.0-10.4). Classifying patients according to the recently developed DIPG survival prediction model, intermediate risk patients (n = 4), showed a PFS and MOS of 6.4 and 12.4 months, respectively, versus a PFS and MOS of 4.5 and 8.1 months, respectively, in high risk patient (n = 5). Gemcitabine up to 200 mg/m2/once weekly, added to radiotherapy, is safe and well tolerated in children with newly diagnosed DIPG. PFS and MOS were not significantly different from literature.

External validation of the diffuse intrinsic pontine glioma survival prediction model: a collaborative report from the International DIPG Registry and the SIOPE DIPG Registry.

We aimed to perform external validation of the recently developed survival prediction model for diffuse intrinsic pontine glioma (DIPG), and discuss its utility. The DIPG survival prediction model was developed in a cohort of patients from the Netherlands, United Kingdom and Germany, registered in the SIOPE DIPG Registry, and includes age <3 years, longer symptom duration and receipt of chemotherapy as favorable predictors, and presence of ring-enhancement on MRI as unfavorable predictor. Model performance was evaluated by analyzing the discrimination and calibration abilities. External validation was performed using an unselected cohort from the International DIPG Registry, including patients from United States, Canada, Australia and New Zealand. Basic comparison with the results of the original study was performed using descriptive statistics, and univariate- and multivariable regression analyses in the validation cohort. External validation was assessed following a variety of analyses described previously. Baseline patient characteristics and results from the regression analyses were largely comparable. Kaplan-Meier curves of the validation cohort reproduced separated groups of standard (n = 39), intermediate (n = 125), and high-risk (n = 78) patients. This discriminative ability was confirmed by similar values for the hazard ratios across these risk groups. The calibration curve in the validation cohort showed a symmetric underestimation of the predicted survival probabilities. In this external validation study, we demonstrate that the DIPG survival prediction model has acceptable cross-cohort calibration and is able to discriminate patients with short, average, and increased survival. We discuss how this clinico-radiological model may serve a useful role in current clinical practice.

The international diffuse intrinsic pontine glioma registry: an infrastructure to accelerate collaborative research for an orphan disease.

Diffuse intrinsic pontine glioma (DIPG), a rare, often fatal childhood brain tumor, remains a major therapeutic challenge. In 2012, investigators, funded by the DIPG Collaborative (a philanthropic partnership among 29 private foundations), launched the International DIPG Registry (IDIPGR) to advance understanding of DIPG. Comprised of comprehensive deidentified but linked clinical, imaging, histopathological, and genomic repositories, the IDIPGR uses standardized case report forms for uniform data collection; serial imaging and histopathology are centrally reviewed by IDIPGR neuro-radiologists and neuro-pathologists, respectively. Tissue and genomic data, and cell cultures derived from autopsies coordinated by the IDIPGR are available to investigators for studies approved by the Scientific Advisory Committee. From April 2012 to December 2016, 670 patients diagnosed with DIPG have been enrolled from 55 participating institutions in the US, Canada, Australia and New Zealand. The radiology repository contains 3558 studies from 448 patients. The pathology repository contains tissue on 81 patients with another 98 samples available for submission. Fresh DIPG tissue from seven autopsies has been sent to investigators to develop primary cell cultures. The bioinformatics repository contains next-generation sequencing data on 66 tumors. Nine projects using data/tissue from the IDIPGR by 13 principle investigators from around the world are now underway. The IDIPGR, a successful alliance among philanthropic agencies and investigators, has developed and maintained a highly collaborative, hypothesis-driven research infrastructure for interdisciplinary and translational projects in DIPG to improve diagnosis, response assessment, treatment and outcome for patients.

State of affairs in use of steroids in diffuse intrinsic pontine glioma: an international survey and a review of the literature.

Children diagnosed with diffuse intrinsic pontine glioma (DIPG) face a dismal prognosis, with severe neurologic deterioration and inevitable death at a median of 9 months from diagnosis. Steroids are widely prescribed as supportive or palliative treatment although they are known to cause severe side effects that may reduce the quality of life. This study aims to review the current knowledge on, and use of, steroids in DIPG patients. A global questionnaire-study among health care professionals was performed to ascertain information on the current (multi-)institutional and (multi-)national use of steroids, the availability of clinical guidelines, and the need for improvements in prescribing steroids to DIPG patients. In addition, an extensive literature search was performed to review studies investigating steroids in pediatric brain tumor patients. From 150 responding health care professionals, only 7 % had clinical guidelines. The use of steroids was heterogeneous and over 85 % of respondents reported serious side effects. Fourteen articles, with low level of evidence, described the use of steroids in pediatric brain tumor patients. Clinical trials investigating optimal dose or regimen were lacking. This study is a first inventory of the availability of evidence-based information and clinical guidelines, and the current attitude towards the use of steroids in DIPG patients. To date, the risk-benefit ratio of steroids in this disease is yet to be determined. We emphasize the need for clinical trials resulting in guidelines on steroids, and possibly alternative drugs, to optimize the quality of care and quality of life of DIPG patients.

Psychosocial profile of pediatric brain tumor survivors with neurocognitive complaints.

PURPOSE: With more children surviving a brain tumor, neurocognitive consequences of the tumor and its treatment become apparent, which could affect psychosocial functioning. The present study therefore aimed to assess psychosocial functioning of pediatric brain tumor survivors (PBTS) in detail. METHODS: Psychosocial functioning of PBTS (8-18 years) with parent-reported neurocognitive complaints was compared to normative data on health-related quality of life (HRQOL), self-esteem, psychosocial adjustment, and executive functioning (one-sample t tests) and to a sibling control group on fatigue (independent-samples t test). Self-, parent-, and teacher-report questionnaires were included, where appropriate, providing complementary information. RESULTS: Eighty-two PBTS (mean age 13.4 years, SD 3.2, 49 % males) and 43 healthy siblings (mean age 14.3, SD 2.4, 40 % males) were included. As compared to the normative population, PBTS themselves reported decreased physical, psychological, and generic HRQOL (d = 0.39-0.62, p < 0.008). Compared to siblings, increased fatigue-related concentration problems (d = 0.57, p < 0.01) were reported, although self-reported self-esteem and psychosocial adjustment seemed not to be affected. Parents of PBTS reported more psychosocial (d = 0.81, p < 0.000) and executive problems (d = 0.35-0.43, p < 0.016) in their child than parents of children in the normative population. Teachers indicated more psychosocial adjustment problems for female PBTS aged 8-11 years than for the female normative population (d = 0.69, p < 0.025), but they reported no more executive problems. CONCLUSIONS: PBTS with parent-reported neurocognitive complaints showed increased psychosocial problems, as reported by PBTS, parents, and teachers. IMPLICATIONS FOR CANCER SURVIVORS: Systematic screening of psychosocial functioning is necessary so that tailored support from professionals can be offered to PBTS with neurocognitive complaints.

Human pontine glioma cells can induce murine tumors.

Diffuse intrinsic pontine glioma (DIPG), with a median survival of only 9 months, is the leading cause of pediatric brain cancer mortality. Dearth of tumor tissue for research has limited progress in this disease until recently. New experimental models for DIPG research are now emerging. To develop preclinical models of DIPG, two different methods were adopted: cells obtained at autopsy (1) were directly xenografted orthotopically into the pons of immunodeficient mice without an intervening cell culture step or (2) were first cultured in vitro and, upon successful expansion, injected in vivo. Both strategies resulted in pontine tumors histopathologically similar to the original human DIPG tumors. However, following the direct transplantation method all tumors proved to be composed of murine and not of human cells. This is in contrast to the indirect method that included initial in vitro culture and resulted in xenografts comprising human cells. Of note, direct injection of cells obtained postmortem from the pons and frontal lobe of human brains not affected by cancer did not give rise to neoplasms. The murine pontine tumors exhibited an immunophenotype similar to human DIPG, but were also positive for microglia/macrophage markers, such as CD45, CD68 and CD11b. Serial orthotopic injection of these murine cells results in lethal tumors in recipient mice. Direct injection of human DIPG cells in vivo can give rise to malignant murine tumors. This represents an important caveat for xenotransplantation models of DIPG. In contrast, an initial in vitro culture step can allow establishment of human orthotopic xenografts. The mechanism underlying this phenomenon observed with direct xenotransplantation remains an open question.

Focused Ultrasound-Enhanced Liquid Biopsy: A Promising Diagnostic Tool for Brain Tumor Patients.

The performance of minimally invasive molecular diagnostic tools in brain tumors, such as liquid biopsy, has so far been limited by the blood-brain barrier (BBB). The BBB hinders the release of brain tumor biomarkers into the bloodstream. The use of focused ultrasound in conjunction with microbubbles has been shown to temporarily open the BBB (FUS-BBBO). This may enhance blood-based tumor biomarker levels. This systematic review provides an overview of the data regarding FUS-BBBO-enhanced liquid biopsy for primary brain tumors. A systematic search was conducted in PubMed and Embase databases with key terms "brain tumors", "liquid biopsy", "FUS" and their synonyms, in accordance with PRISMA statement guidelines. Five preclinical and two clinical studies were included. Preclinical studies utilized mouse, rat and porcine glioma models. Biomarker levels were found to be higher in sonicated groups compared to control groups. Both stable and inertial microbubble cavitation increased biomarker levels, whereas only inertial cavitation induced microhemorrhages. In clinical studies involving 14 patients with high-grade brain tumors, biomarker levels were increased after FUS-BBBO with stable cavitation. In conclusion, FUS-BBBO-enhanced liquid biopsy using stable cavitation shows diagnostic potential for primary brain tumors. Further research is imperative before integrating FUS-BBBO for liquid biopsy enhancement into clinical practice.