Reversible Addition-Fragmentation Chain-Transfer Polymerization in Supercritical CO2: A Review.

The development of cleaner, more environmentally friendly processes in polymerization technology is crucial due to the prevalent use of volatile organic solvents (VOCs), which are harmful and toxic. Future regulations are likely to limit or ban VOCs. This review explores the use of supercritical solvents, specifically supercritical CO2 (scCO2), in polymerization processes. The study focuses on reversible addition-fragmentation chain-transfer (RAFT) induced homo-polymerization of various monomers using specific chain transfer agents (CTAs) in scCO2. RAFT polymerization, a reversible deactivation radical polymerization (RDRP) polymerization, relies heavily on the choice of CTA, which significantly influences the dispersity and molar mass of the resulting polymers. Stabilizers are also crucial in controlling product specifications for polymerizations in supercritical CO2, except for fluor-based polymers, although they must be removed and preferably recycled to ensure product purity and sustainability. The review notes that achieving high molar mass through RAFT polymerization in scCO2 is challenging due to solubility limits, which lead to polymer precipitation. Despite this, RAFT polymerization in scCO2 shows promise for sustainable, circular production of low molar mass polymers, although these cannot yet be fully considered green products.

The heterogeneous sensitivity of pediatric brain tumors to different oncolytic viruses is predicted by unique gene expression profiles.

Despite decades of research, the prognosis of high-grade pediatric brain tumors (PBTs) remains dismal; however, recent cases of favorable clinical responses were documented in clinical trials using oncolytic viruses (OVs). In the current study, we employed four different species of OVs: adenovirus Delta24-RGD, herpes simplex virus rQNestin34.5v1, reovirus R124, and the non-virulent Newcastle disease virus rNDV-F0-GFP against three entities of PBTs (high-grade gliomas, atypical teratoid/rhabdoid tumors, and ependymomas) to determine their in vitro efficacy. These four OVs were screened on 14 patient-derived PBT cell cultures and the degree of oncolysis was assessed using an ATP-based assay. Subsequently, the observed viral efficacies were correlated to whole transcriptome data and Gene Ontology analysis was performed. Although no significant tumor type-specific OV efficacy was observed, the analysis revealed the intrinsic biological processes that associated with OV efficacy. The predictive power of the identified expression profiles was further validated in vitro by screening additional PBTs. In summary, our results demonstrate OV susceptibility of multiple patient-derived PBT entities and the ability to predict in vitro responses to OVs using unique expression profiles. Such profiles may hold promise for future OV preselection with effective oncolytic potency in a specific tumor, therewith potentially improving OV responses.

OV Modulators of the Paediatric Brain TIME: Current Status, Combination Strategies, Limitations and Future Directions.

Oncolytic viruses (OVs) are characterised by their preference for infecting and replicating in tumour cells either naturally or after genetic modification, resulting in oncolysis. Furthermore, OVs can elicit both local and systemic anticancer immune responses while specifically infecting and lysing tumour cells. These characteristics render them a promising therapeutic approach for paediatric brain tumours (PBTs). PBTs are frequently marked by a cold tumour immune microenvironment (TIME), which suppresses immunotherapies. Recent preclinical and clinical studies have demonstrated the capability of OVs to induce a proinflammatory immune response, thereby modifying the TIME. In-depth insights into the effect of OVs on different cell types in the TIME may therefore provide a compelling basis for using OVs in combination with other immunotherapy modalities. However, certain limitations persist in our understanding of oncolytic viruses' ability to regulate the TIME to enhance anti-tumour activity. These limitations primarily stem from the translational limitations of model systems, the difficulties associated with tracking reliable markers of efficacy throughout the course of treatment and the role of pre-existing viral immunity. In this review, we describe the different alterations observed in the TIME in PBTs due to OV treatment, combination therapies of OVs with different immunotherapies and the hurdles limiting the development of effective OV therapies while suggesting future directions based on existing evidence.

BEHAV3D: a 3D live imaging platform for comprehensive analysis of engineered T cell behavior and tumor response.

Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results. Together, this enables a refined assessment of how solid and liquid tumors respond to cellular immunotherapy, critically capturing both inter- and intratumoral heterogeneity in treatment response. In addition, BEHAV3D uncovers T cell behavior involved in tumor targeting, offering insight into their mode of action. Our pipeline thereby has strong implications for comparing, prioritizing and improving immunotherapy products by highlighting the behavioral differences between individual tumor donors, distinct T cell therapy concepts or subpopulations. The protocol describes critical wet lab steps, including co-culture preparations and fast 3D imaging with live cell dyes, a segmentation-based image processing tool to track individual organoids, tumor and immune cells and an analytical pipeline for behavioral profiling. This 1-week protocol, accessible to users with basic cell culture, imaging and programming expertise, can easily be adapted to any type of co-culture to visualize and exploit cell behavior, having far-reaching implications for the immuno-oncology field and beyond.

INSPIRED Symposium Part 4B: Chimeric Antigen Receptor T Cell Correlative Studies-Established Findings and Future Priorities.

Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies, with multiple CAR T cell products approved for numerous indications by regulatory agencies worldwide. However, significant work remains to be done to enhance these treatments. In March 2023, a group of experts in CAR T cell therapy assembled at the National Institutes of Health in Bethesda, Maryland at the Insights in Pediatric CAR T Cell Immunotherapy: Recent Advances and Future Directions (INSPIRED) Symposium to identify key areas for research for the coming years. In session 4B, correlative studies to be incorporated into future clinical trials and real-world settings were discussed. Active areas of research identified included (1) optimizing CAR T cell product manufacturing; (2) ensuring adequate lymphodepletion prior to CAR T cell administration; (3) overcoming immunoregulatory cells and tumor stroma present in the tumor microenvironment, particularly in solid tumors; (4) understanding tumor intrinsic properties that lead to CAR T cell immunotherapy resistance; and (5) uncovering biomarkers predictive of treatment resistance, treatment durability, or immune-related adverse events. Here we review the results of previously published clinical trials and real-world studies to summarize what is currently known about each of these topics. We then outline priorities for future research that we believe will be important for improving our understanding of CAR T cell therapy and ultimately leading to better outcomes for patients.

Deep Learning for Detection and Localization of B-Lines in Lung Ultrasound.

Lung ultrasound (LUS) is an important imaging modality used by emergency physicians to assess pulmonary congestion at the patient bedside. B-line artifacts in LUS videos are key findings associated with pulmonary congestion. Not only can the interpretation of LUS be challenging for novice operators, but visual quantification of B-lines remains subject to observer variability. In this work, we investigate the strengths and weaknesses of multiple deep learning approaches for automated B-line detection and localization in LUS videos. We curate and publish, BEDLUS, a new ultrasound dataset comprising 1,419 videos from 113 patients with a total of 15,755 expert-annotated B-lines. Based on this dataset, we present a benchmark of established deep learning methods applied to the task of B-line detection. To pave the way for interpretable quantification of B-lines, we propose a novel "single-point" approach to B-line localization using only the point of origin. Our results show that (a) the area under the receiver operating characteristic curve ranges from 0.864 to 0.955 for the benchmarked detection methods, (b) within this range, the best performance is achieved by models that leverage multiple successive frames as input, and (c) the proposed single-point approach for B-line localization reaches an F 1-score of 0.65, performing on par with the inter-observer agreement. The dataset and developed methods can facilitate further biomedical research on automated interpretation of lung ultrasound with the potential to expand the clinical utility.

Automated production of specific T cells for treatment of refractory viral infections after allogeneic stem cell transplantation.

Therapy-resistant viral reactivations contribute significantly to mortality after hematopoietic stem cell transplantation. Adoptive cellular therapy with virus-specific T cells (VST) has shown efficacy in various single-center trials. However, the scalability of this therapy is hampered by laborious production methods. In this study we describe the in-house production of VST in a closed system (CliniMACS Prodigy® system, Miltenyi Biotec). In addition, we report the efficacy in 26 patients with viral disease following hematopoietic stem cell transplantation in a retrospective analysis (adenovirus, n=7; cytomegalovirus, n=8; Epstein-Barr virus, n=4; multi-viral, n=7). The production of VST was successful in 100% of cases. The safety profile of VST therapy was favorable (n=2 grade 3 and n=1 grade 4 adverse events; all three were reversible). A response was seen in 20 of 26 patients (77%). Responding patients had a significantly better overall survival than patients who did not respond (P<0.001). Virus-specific symptoms were reduced or resolved in 47% of patients. The overall survival of the whole cohort was 28% after 6 months. This study shows the feasibility of automated VST production and safety of application. The scalability of the CliniMACS Prodigy® device increases the accessibility of VST treatment.

SARS-CoV-2 infections in pediatric and young adult recipients of chimeric antigen receptor T-cell therapy: an international registry report.

BACKGROUND: Immunocompromised patients are at increased risk of SARS-CoV-2 infections. Patients undergoing chimeric antigen receptor (CAR) T-cell therapy for relapsed/refractory B-cell malignancies are uniquely immunosuppressed due to CAR T-mediated B-cell aplasia (BCA). While SARS-CoV-2 mortality rates of 33%-40% are reported in adult CAR T-cell recipients, outcomes in pediatric and young adult CAR T-cell recipients are limited. METHODS: We created an international retrospective registry of CAR T recipients aged 0-30 years infected with SARS-CoV-2 within 2 months prior to or any time after CAR T infusion. SARS-CoV-2-associated illness was graded as asymptomatic, mild, moderate, or severe COVID-19, or multisystem inflammatory syndrome in children (MIS-C). To assess for risk factors associated with significant SARS-CoV-2 infections (infections requiring hospital admission for respiratory distress or supplemental oxygen), univariate and multivariable regression analyses were performed. RESULTS: Nine centers contributed 78 infections in 75 patients. Of 70 SARS-CoV-2 infections occurring after CAR T infusion, 13 (18.6%) were classified as asymptomatic, 37 (52.9%) mild, 11 (15.7%) moderate, and 6 (8.6%) severe COVID-19. Three (4.3%) were classified as MIS-C. BCA was not significantly associated with infection severity. Prior to the emergence of the Omicron variant, of 47 infections, 19 (40.4%) resulted in hospital admission and 7 (14.9%) required intensive care, while after the emergence of the Omicron variant, of 23 infections, only 1 (4.3%) required admission and the remaining 22 (95.7%) had asymptomatic or mild COVID-19. Death occurred in 3 of 70 (4.3%); each death involved coinfection or life-threatening condition. In a multivariable model, factors associated with significant SARS-CoV-2 infection included having two or more comorbidities (OR 7.73, CI 1.05 to 74.8, p=0.048) and age ≥18 years (OR 9.51, CI 1.90 to 82.2, p=0.014). In the eight patients infected with SARS-CoV-2 before CAR T, half of these patients had their CAR T infusion delayed by 15-30 days. CONCLUSIONS: In a large international cohort of pediatric and young adult CAR-T recipients, SARS-CoV-2 infections resulted in frequent hospital and intensive care unit admissions and were associated with mortality in 4.3%. Patients with two or more comorbidities or aged ≥18 years were more likely to experience significant illness. Suspected Omicron infections were associated with milder disease.

Outcome of chimeric antigen receptor T-cell therapy following treatment with inotuzumab ozogamicin in children with relapsed or refractory acute lymphoblastic leukemia.

Chimeric antigen receptor T cells targeting CD19 (CART-19) have shown remarkable efficacy for relapsed/refractory (R/R) B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We investigated whether prior use of inotuzumab ozogamicin (InO), an anti-CD22 antibody conjugated to calicheamicin, may impact CAR T-cell manufacturing or efficacy via pre-CART-19 depletion of the B-cell compartment. In this international, retrospective analysis, 39 children and young adults receiving InO before (n = 12) and/or after (n = 27) T-cell apheresis as bridging therapy to CART-19 treatment were analyzed. Median age at infusion was 13 years (range 1.4-23 years). Thirty-four out of 39 patients (87.2%) obtained complete remission. With a median follow-up of 18.2 months after CART-19 infusion, 12-month event-free survival (EFS) was 53.3% (95% confidence interval (CI): 38.7-73.4) and overall survival (OS) was 77.8% (95% CI: 64.5-93.9). Seventeen patients (44%) relapsed with a median of 159 days (range 28-655) after CART-19 infusion. No difference in day 28 minimal residual disease negative complete response rate, 12-month OS/EFS, or incidence of CD19-positive or -negative relapses was observed among patients receiving InO before or after apheresis. Compared to published data for patients treated with CART-19 therapy without prior InO exposure, response and OS/EFS for patients treated with InO prior to CART-19 are similar.

Automatic evaluation of graft orientation during Descemet membrane endothelial keratoplasty using intraoperative OCT.

Correct Descemet Membrane Endothelial Keratoplasty (DMEK) graft orientation is imperative for success of DMEK surgery, but intraoperative evaluation can be challenging. We present a method for automatic evaluation of the graft orientation in intraoperative optical coherence tomography (iOCT), exploiting the natural rolling behavior of the graft. The method encompasses a deep learning model for graft segmentation, post-processing to obtain a smooth line representation, and curvature calculations to determine graft orientation. For an independent test set of 100 iOCT-frames, the automatic method correctly identified graft orientation in 78 frames and obtained an area under the receiver operating characteristic curve (AUC) of 0.84. When we replaced the automatic segmentation with the manual masks, the AUC increased to 0.92, corresponding to an accuracy of 86%. In comparison, two corneal specialists correctly identified graft orientation in 90% and 91% of the iOCT-frames.

Knowledge distillation with ensembles of convolutional neural networks for medical image segmentation.

Purpose: Ensembles of convolutional neural networks (CNNs) often outperform a single CNN in medical image segmentation tasks, but inference is computationally more expensive and makes ensembles unattractive for some applications. We compared the performance of differently constructed ensembles with the performance of CNNs derived from these ensembles using knowledge distillation, a technique for reducing the footprint of large models such as ensembles. Approach: We investigated two different types of ensembles, namely, diverse ensembles of networks with three different architectures and two different loss-functions, and uniform ensembles of networks with the same architecture but initialized with different random seeds. For each ensemble, additionally, a single student network was trained to mimic the class probabilities predicted by the teacher model, the ensemble. We evaluated the performance of each network, the ensembles, and the corresponding distilled networks across three different publicly available datasets. These included chest computed tomography scans with four annotated organs of interest, brain magnetic resonance imaging (MRI) with six annotated brain structures, and cardiac cine-MRI with three annotated heart structures. Results: Both uniform and diverse ensembles obtained better results than any of the individual networks in the ensemble. Furthermore, applying knowledge distillation resulted in a single network that was smaller and faster without compromising performance compared with the ensemble it learned from. The distilled networks significantly outperformed the same network trained with reference segmentation instead of knowledge distillation. Conclusion: Knowledge distillation can compress segmentation ensembles of uniform or diverse composition into a single CNN while maintaining the performance of the ensemble.

RAFT Polymerization of a Biorenewable/Sustainable Monomer via a Green Process.

A biorenewable polymer is synthesized via a green process using the RAFT principle for the first time in supercritical CO2 at 300 bar and 80 °C. α-Methylene-γ-butyrolactone polymers of various chain lengths and molecular weights are obtained. The molecular weights vary from 10 000 up to 20 000 with low polydispersity indexes (PDI <1.5). Furthermore, the monomer conversion in supercritical CO2 is substantially higher, respectively 85% for ScCO2 compared to ≈65% for polymerizations conducted in dimethyl formamide (DMF) solvent. Chain extensions are carried out to confirm the livingness of the formed polymers in ScCO2 . This opens up future possibilities of the formation of different polymer architectures in ScCO2 . The polymers synthesized in ScCO2 have glass transition temperature (Tg ) values ranging from 155 up to 190 °C. However, the presence of residual monomer encapsulated inside the formed polymer matrix affects the glass transition of the polymer that is lowered by increasing monomer concentrations. Hence, additional research is required to eliminate the remaining monomer concentration in the polymer matrix in order to arrive at the optimal Tg .

A Toolkit for Profiling the Immune Landscape of Pediatric Central Nervous System Malignancies.

The prognosis of pediatric central nervous system (CNS) malignancies remains dismal due to limited treatment options, resulting in high mortality rates and long-term morbidities. Immunotherapies, including checkpoint inhibition, cancer vaccines, engineered T cell therapies, and oncolytic viruses, have promising results in some hematological and solid malignancies, and are being investigated in clinical trials for various high-grade CNS malignancies. However, the role of the tumor immune microenvironment (TIME) in CNS malignancies is mostly unknown for pediatric cases. In order to successfully implement immunotherapies and to eventually predict which patients would benefit from such treatments, in-depth characterization of the TIME at diagnosis and throughout treatment is essential. In this review, we provide an overview of techniques for immune profiling of CNS malignancies, and detail how they can be utilized for different tissue types and studies. These techniques include immunohistochemistry and flow cytometry for quantifying and phenotyping the infiltrating immune cells, bulk and single-cell transcriptomics for describing the implicated immunological pathways, as well as functional assays. Finally, we aim to describe the potential benefits of evaluating other compartments of the immune system implicated by cancer therapies, such as cerebrospinal fluid and blood, and how such liquid biopsies are informative when designing immune monitoring studies. Understanding and uniformly evaluating the TIME and immune landscape of pediatric CNS malignancies will be essential to eventually integrate immunotherapy into clinical practice.

Fludarabine exposure predicts outcome after CD19 CAR T-cell therapy in children and young adults with acute leukemia.

The addition of fludarabine to cyclophosphamide as a lymphodepleting regimen prior to CD19 chimeric antigen receptor (CAR) T-cell therapy significantly improved outcomes in patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). Fludarabine exposure, previously shown to be highly variable when dosing is based on body surface area (BSA), is a predictor for survival in allogeneic hematopoietic cell transplantation (allo-HCT). Hence, we hypothesized that an optimal exposure of fludarabine might be of clinical importance in CD19 CAR T-cell treatment. We examined the effect of cumulative fludarabine exposure during lymphodepletion, defined as concentration-time curve (AUC), on clinical outcome and lymphocyte kinetics. A retrospective analysis was conducted with data from 26 patients receiving tisagenlecleucel for r/r B-ALL. Exposure of fludarabine was shown to be a predictor for leukemia-free survival (LFS), B-cell aplasia, and CD19-positive relapse following CAR T-cell infusion. Minimal event probability was observed at a cumulative fludarabine AUCT0-∞ ≥14 mg*h/L, and underexposure was defined as an AUCT0-∞ <14 mg*h/L. In the underexposed group, the median LFS was 1.8 months, and the occurrence of CD19-positive relapse within 1 year was 100%, which was higher compared with the group with an AUCT0-∞ ≥14 mg*h/L (12.9 months; P < .001; and 27.4%; P = .0001, respectively). Furthermore, the duration of B-cell aplasia within 6 months was shorter in the underexposed group (77.3% vs 37.3%; P = .009). These results suggest that optimizing fludarabine exposure may have a relevant impact on LFS following CAR T-cell therapy, which needs to be validated in a prospective clinical trial.

Paving the Way for Immunotherapy in Pediatric Acute Myeloid Leukemia: Current Knowledge and the Way Forward.

Immunotherapeutic agents may be an attractive option to further improve outcomes and to reduce treatment-related toxicity for pediatric AML. While improvements in outcome have been observed with immunotherapy in many cancer types, immunotherapy development and implementation into patient care for both adult and pediatric AML has been hampered by an incomplete understanding of the bone marrow environment and a paucity of tumor-specific antigens. Since only a minority of patients respond in most immunotherapy trials across different cancer types, it will be crucial to understand which children with AML are likely to respond to or may benefit from immunotherapies. Immune cell profiling efforts hold promise to answer this question, as illustrated by the development of predictive scores in solid cancers. Such information on the number and phenotype of immune cells during current treatment regimens will be pivotal to generate hypotheses on how and when to intervene with immunotherapy in pediatric AML. In this review, we discuss the current understanding of the number and phenotype of immune cells in the bone marrow in pediatric AML, ongoing immunotherapy trials and how comprehensive immune profiling efforts may pave the way for successful clinical trials (and, ultimately, implementation into patient care).

Comparing the signal enhancement of a gadolinium based and an iron-oxide based contrast agent in low-field MRI.

Recently, there has been a renewed interest in low-field MRI. Contrast agents (CA) in MRI have magnetic behavior dependent on magnetic field strength. Therefore, the optimal contrast agent for low-field MRI might be different from what is used at higher fields. Ultra-small superparamagnetic iron-oxides (USPIOs), commonly used as negative CA, might also be used for generating positive contrast in low-field MRI. The purpose of this study was to determine whether an USPIO or a gadolinium based contrast agent is more appropriate at low field strengths. Relaxivity values of ferumoxytol (USPIO) and gadoterate (gadolinium based) were used in this research to simulate normalized signal intensity (SI) curves within a concentration range of 0-15 mM. Simulations were experimentally validated on a 0.25T MRI scanner. Simulations and experiments were performed using spin echo (SE), spoiled gradient echo (SGE), and balanced steady-state free precession (bSSFP) sequences. Maximum achievable SIs were assessed for both CAs in a range of concentrations on all sequences. Simulations at 0.25T showed a peak in SIs at low concentrations ferumoxytol versus a wide top at higher concentrations for gadoterate in SE and SGE. Experiments agreed well with the simulations in SE and SGE, but less in the bSSFP sequence due to overestimated relaxivities in simulations. At low magnetic field strengths, ferumoxytol generates similar signal enhancement at lower concentrations than gadoterate.

Corneal pachymetry by AS-OCT after Descemet's membrane endothelial keratoplasty.

Corneal thickness (pachymetry) maps can be used to monitor restoration of corneal endothelial function, for example after Descemet's membrane endothelial keratoplasty (DMEK). Automated delineation of the corneal interfaces in anterior segment optical coherence tomography (AS-OCT) can be challenging for corneas that are irregularly shaped due to pathology, or as a consequence of surgery, leading to incorrect thickness measurements. In this research, deep learning is used to automatically delineate the corneal interfaces and measure corneal thickness with high accuracy in post-DMEK AS-OCT B-scans. Three different deep learning strategies were developed based on 960 B-scans from 50 patients. On an independent test set of 320 B-scans, corneal thickness could be measured with an error of 13.98 to 15.50 µm for the central 9 mm range, which is less than 3% of the average corneal thickness. The accurate thickness measurements were used to construct detailed pachymetry maps. Moreover, follow-up scans could be registered based on anatomical landmarks to obtain differential pachymetry maps. These maps may enable a more comprehensive understanding of the restoration of the endothelial function after DMEK, where thickness often varies throughout different regions of the cornea, and subsequently contribute to a standardized postoperative regime.

Chimeric Antigen Receptor T-Cell Therapy in Paediatric B-Cell Precursor Acute Lymphoblastic Leukaemia: Curative Treatment Option or Bridge to Transplant?

Chimeric antigen receptor T-cell therapy (CAR-T) targeting CD19 has been associated with remarkable responses in paediatric patients and adolescents and young adults (AYA) with relapsed/refractory (R/R) B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). Tisagenlecleucel, the first approved CD19 CAR-T, has become a viable treatment option for paediatric patients and AYAs with BCP-ALL relapsing repeatedly or after haematopoietic stem cell transplantation (HSCT). Based on the chimeric antigen receptor molecular design and the presence of a 4-1BB costimulatory domain, tisagenlecleucel can persist for a long time and thereby provide sustained leukaemia control. "Real-world" experience with tisagenlecleucel confirms the safety and efficacy profile observed in the pivotal registration trial. Recent guidelines for the recognition, management and prevention of the two most common adverse events related to CAR-T - cytokine release syndrome and immune-cell-associated neurotoxicity syndrome - have helped to further decrease treatment toxicity. Consequently, the questions of how and for whom CD19 CAR-T could substitute HSCT in BCP-ALL are inevitable. Currently, 40-50% of R/R BCP-ALL patients relapse post CD19 CAR-T with either CD19- or CD19+ disease, and consolidative HSCT has been proposed to avoid disease recurrence. Contrarily, CD19 CAR-T is currently being investigated in the upfront treatment of high-risk BCP-ALL with an aim to avoid allogeneic HSCT and associated treatment-related morbidity, mortality and late effects. To improve survival and decrease long-term side effects in children with BCP-ALL, it is important to define parameters predicting the success or failure of CAR-T, allowing the careful selection of candidates in need of HSCT consolidation. In this review, we describe the current clinical evidence on CAR-T in BCP-ALL and discuss factors associated with response to or failure of this therapy: product specifications, patient- and disease-related factors and the impact of additional therapies given before (e.g., blinatumomab and inotuzumab ozogamicin) or after infusion (e.g., CAR-T re-infusion and/or checkpoint inhibition). We discuss where to position CAR-T in the treatment of BCP-ALL and present considerations for the design of supportive trials for the different phases of disease. Finally, we elaborate on clinical settings in which CAR-T might indeed replace HSCT.