Fully equipped CARs to address tumor heterogeneity, enhance safety, and improve the functionality of cellular immunotherapies.

Although adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells has achieved unprecedented response rates in patients with certain hematological malignancies, this therapeutic modality is still far from fulfilling its remarkable potential, especially in the context of solid cancers. Antigen escape variants, off-tumor destruction of healthy tissues expressing tumor-associated antigens (TAAs), poor CAR-T cell persistence, and the occurrence of functional exhaustion represent some of the most prominent hurdles that limit CAR-T cell ability to induce long-lasting remissions with a tolerable adverse effect profile. In this review, we summarize the main approaches that have been developed to face such bottlenecks, including the adapter CAR (AdCAR) system, Boolean-logic gating, epitope editing, the modulation of cell-intrinsic signaling pathways, and the incorporation of safety switches to precisely control CAR-T cell activation. We also discuss the most pressing issues pertaining to the selection of co-stimulatory domains, with a focus on strategies aimed at promoting CAR-T cell persistence and optimal antitumor functionality.

Perioperative Care for Children With Syndromic Craniofacial Synostosis Undergoing Le Fort III Surgery: A Retrospective Cohort Study.

OBJECTIVE: To present characteristics, surgical variables, complications, and postoperative care in pediatric patients with craniofacial synostosis undergoing Le Fort III osteotomy. BACKGROUND: Craniofacial synostoses are a group of genetic syndromes that result in premature fusion of cranial and facial sutures, leading to craniofacial deformities and associated complications. Midface advancement through Le Fort III osteotomy is the most frequent surgical option for these conditions. METHODS: Retrospective monocentric cohort study including patients with syndromic craniofacial synostosis who underwent Le Fort III osteotomy between 2009 and 2022 in a specialized referral center. Data collection encompassed surgical time, blood loss, intraoperative transfusions, fluid balance, and postoperative parameters such as duration of invasive mechanical ventilation and intensive care unit (ICU) length of stay. RESULTS: Twenty-six children were included in the analysis. The median surgical time was 345 minutes (300-360), with an estimated blood loss of 15 (9.9-24) mL/kg. Patients required a median transfusion of 12.63 (7.1-24.5) mL/kg of packed red blood cells and 19.82 (11.1-33) mL/kg of fresh frozen plasma. Intraoperative fluid balance was + 12.5 (0.8-22.8) mL/kg, with a median infusion of 30.4 (23.9-38.7) mL/kg of crystalloids. All patients were transferred to the ICU after surgery to ensure a safe environment for extubation. The median duration of mechanical ventilation in the ICU was 30 (20.25-45) hours, and postoperative ICU length of stay was 2 (2-4) days, and complications were infrequent, with only one extubation failure recorded. CONCLUSION: Le Fort III osteotomy in craniofacial synostosis patients may be characterized by a complex perioperative course. A multidisciplinary approach in the care of these patients allows for minimizing complications in the perioperative phase. Further research is needed to enhance perioperative management in this unique patient population.

ETV6::RUNX1 Acute Lymphoblastic Leukemia: how much therapy is needed for cure?

Recent trials show 5-year survival rates >95% for ETV6::RUNX1 Acute Lymphoblastic Leukemia (ALL). Since treatment has many side effects, an overview of cumulative drug doses and intensities between eight international trials is presented to characterize therapy needed for cure. A meta-analysis was performed as a comprehensive summary of survival outcomes at 5 and 10 years. For drug dose comparison in non-high risk trial arms, risk group distribution was applied to split the trials into two groups: trial group A with ~70% (range: 63.5-75%) of patients in low risk (LR) (CCLSG ALL2004, CoALL 07-03, NOPHO ALL2008, UKALL2003) and trial group B with ~45% (range: 38.7-52.7%) in LR (AIEOP-BFM ALL 2000, ALL-IC BFM ALL 2002, DCOG ALL10, JACLS ALL-02). Meta-analysis did not show evidence of heterogeneity between studies in trial group A LR and medium risk (MR) despite differences in treatment intensity. Statistical heterogeneity was present in trial group B LR and MR. Trials using higher cumulative dose and intensity of asparaginase and pulses of glucocorticoids and vincristine showed better 5-year event-free survival but similar overall survival. Based on similar outcomes between trials despite differences in therapy intensity, future trials should investigate, to what extent de-escalation is feasible for ETV6::RUNX1 ALL.

Safety and efficacy of narsoplimab in pediatric and adult patients with transplant-associated thrombotic microangiopathy: a real-world experience.

Transplant-associated thrombotic microangiopathy (TA-TMA) is a severe complication following hematopoietic stem cell transplantation (HSCT). No approved treatments are currently available. This study presents real-world data obtained with narsoplimab, a human immunoglobulin G4 monoclonal antibody that inhibits MASP-2, the effector enzyme of the lectin pathway of the complement system. Between January 2018 and August 2023, 20 (13 adult and 7 pediatric) patients diagnosed with TA-TMA received narsoplimab under an ongoing compassionate use program. The diagnosis was based on internationally defined criteria for pediatric and adult patients. Fifteen patients fulfilled the criteria recently established by an international consensus on TA-TMA. Nineteen patients exhibited high-risk characteristics. Thirteen patients (65%) responded to narsoplimab, achieving transfusion independence and significant clinical improvement. The one-hundred-day Overall Survival (OS) post-TA-TMA diagnosis was 70%, and 100% for responders. Narsoplimab proved to be effective and safe in the treatment of high-risk TA-TMA, with no increased infectious complications or other safety signals of concern across all age groups. The high response rates and the encouraging survival outcomes underscore the potential of narsoplimab as a valuable therapeutic option, particularly for high-risk cases.

Multimodal wearable EEG, EMG and accelerometry measurements improve the accuracy of tonic-clonic seizure detection.

Objective. This paper aims to investigate the possibility of detecting tonic-clonic seizures (TCSs) with behind-the-ear, two-channel wearable electroencephalography (EEG), and to evaluate its added value to non-EEG modalities in TCS detection.Methods. We included 27 participants with a total of 44 TCSs from the European multicenter study SeizeIT2. The wearable Sensor Dot (Byteflies) was used to measure behind-the-ear EEG, electromyography (EMG), electrocardiography, accelerometry (ACC) and gyroscope. We evaluated automatic unimodal detection of TCSs, using sensitivity, precision, false positive rate (FPR) and F1-score. Subsequently, we fused the different modalities and again assessed performance. Algorithm-labeled segments were then provided to two experts, who annotated true positive TCSs, and discarded false positives.Results. Wearable EEG outperformed the other single modalities with a sensitivity of 100% and a FPR of 10.3/24 h. The combination of wearable EEG and EMG proved most clinically useful, delivering a sensitivity of 97.7%, an FPR of 0.4/24 h, a precision of 43%, and an F1-score of 59.7%. The highest overall performance was achieved through the fusion of wearable EEG, EMG, and ACC, yielding a sensitivity of 90.9%, an FPR of 0.1/24 h, a precision of 75.5%, and an F1-score of 82.5%.Conclusions. In TCS detection with a wearable device, combining EEG with EMG, ACC or both resulted in a remarkable reduction of FPR, while retaining a high sensitivity.Significance. Adding wearable EEG could further improve TCS detection, relative to extracerebral-based systems.

Endocrine, auxological and metabolic profile in children and adolescents with Down syndrome: from infancy to the first steps into adult life.

Down syndrome (DS) is the most common chromosomal disorder worldwide. Along with intellectual disability, endocrine disorders represent a remarkable share of the morbidities experienced by children, adolescents and young adults with DS. Auxological parameters are plotted on syndrome-specific charts, as growth rates are reduced compared to healthy age- and gender-matched peers. Furthermore, children with DS are at increased risk for thyroid dysfunctions, diabetes mellitus, osteopenia and obesity compared to general population. Additionally, male individuals with DS often show infertility, while women tend to experience menopause at an overall younger age than healthy controls. Given the recent outstanding improvements in the care of severe DS-related comorbidities, infant mortality has dramatically decreased, with a current average life expectancy exceeding 60 years. Accordingly, the awareness of the specificities of DS in this field is pivotal to timely detect endocrine dysfunctions and to undertake a prompt dedicated treatment. Notably, best practices for the screening and monitoring of pediatric endocrine disorders in DS are still controversial. In addition, specific guidelines for the management of metabolic issues along the challenging period of transitioning from pediatric to adult health care are lacking. By performing a review of published literature, we highlighted the issues specifically involving children and adolescent with DS, aiming at providing clinicians with a detailed up-to-date overview of the endocrine, metabolic and auxological disorders in this selected population, with an additional focus on the management of patients in the critical phase of the transitioning from childhood to adult care.

Healthcare migration in Italian paediatric haematology-oncology centres belonging to AIEOP.

BACKGROUND: In Italy, there is a network of centres headed by the Italian Association of Pediatric Hematology and Oncology (AIEOP) for the diagnosis and treatment of paediatric cancers on almost the entire national territory. Nevertheless, migration of patients in a hospital located in a region different from that of residence is a widespread habit, sometimes motivated by several reasons. The aim of this paper is to assess the impact of migration of children with cancer to AIEOP centres in order to verify their optimal distribution throughout the national territory. METHODS: To this purpose, we used information on 41,205 registered cancer cases in the database of Mod.1.01 Registry from AIEOP centres, with age of less than 20 years old at diagnosis, diagnosed from 1988 to 2017. Patients' characteristics were analysed and compared using the X2 or Fisher's exact test or Mann-Whitney test, when appropriate. Survival distributions were estimated using the method of Kaplan and Meier, and the log-rank test was used to examine differences among subgroups. RESULTS: Extra-regional migration involved overall 19.5% of cases, ranging from 23.3% (1988-1997) to 16.4% (2008-2017) (p < 0.001). In leukaemias and lymphomas we observed a mean migration of 8.8% overall, lower in the North (1.2%) and Centre (7.8%) compared to the South & Isles (32.3%). In the case of solid tumours, overall migration was 25.7%, with 4.2% in the North, 17.2% in the Centre and 59.6% in the South & Isles. For regions with overall levels of migration higher than the national average, most migration cases opted for AIEOP centres of close or even neighbouring regions. Overall survival at 10 years from diagnosis results 69.9% in migrants vs 78.3% in no migrants (p < 0.001). CONCLUSIONS: There is still a certain amount of domestic migration, the causes of which can be easily identified: migration motivated by a search for high specialization, migration due to lack of local facilities, or regions in which no AIEOP centres are present, which makes migration obligatory. Better coordination between AIEOP centres could help to reduce so-called avoidable migration, but technical and political choices will have to be considered, with the active participation of sector technicians.

Modeling skeletal dysplasia in Hurler syndrome using patient-derived bone marrow osteoprogenitor cells.

Dysostosis multiplex is a major cause of morbidity in Hurler syndrome (mucopolysaccharidosis type IH [MPS IH], OMIM #607014) because currently available therapies have limited success in its prevention and reversion. Unfortunately, the elucidation of skeletal pathogenesis in MPS IH is limited by difficulties in obtaining bone specimens from pediatric patients and poor reproducibility in animal models. Thus, the application of experimental systems that can be used to dissect cellular and molecular mechanisms underlying the skeletal phenotype of MPS IH patients and to identify effective therapies is highly needed. Here, we adopted in vitro/in vivo systems based on patient-derived bone marrow stromal cells to generate cartilaginous pellets and bone rudiments. Interestingly, we observed that heparan sulphate accumulation compromised the remodeling of MPS IH cartilage into other skeletal tissues and other critical aspects of the endochondral ossification process. We also noticed that MPS IH hypertrophic cartilage was characterized by dysregulation of signaling pathways controlling cartilage hypertrophy and fate, extracellular matrix organization, and glycosaminoglycan metabolism. Our study demonstrates that the cartilaginous pellet-based system is a valuable tool to study MPS IH dysostosis and to develop new therapeutic approaches for this hard-to-treat aspect of the disease. Finally, our approach may be applied for modeling other genetic skeletal disorders.

Small-molecule CBP/p300 histone acetyltransferase inhibition mobilizes leukocytes from the bone marrow via the endocrine stress response.

Mutations of the CBP/p300 histone acetyltransferase (HAT) domain can be linked to leukemic transformation in humans, suggestive of a checkpoint of leukocyte compartment sizes. Here, we examined the impact of reversible inhibition of this domain by the small-molecule A485. We found that A485 triggered acute and transient mobilization of leukocytes from the bone marrow into the blood. Leukocyte mobilization by A485 was equally potent as, but mechanistically distinct from, granulocyte colony-stimulating factor (G-CSF), which allowed for additive neutrophil mobilization when both compounds were combined. These effects were maintained in models of leukopenia and conferred augmented host defenses. Mechanistically, activation of the hypothalamus-pituitary-adrenal gland (HPA) axis by A485 relayed shifts in leukocyte distribution through corticotropin-releasing hormone receptor 1 (CRHR1) and adrenocorticotropic hormone (ACTH), but independently of glucocorticoids. Our findings identify a strategy for rapid expansion of the blood leukocyte compartment via a neuroendocrine loop, with implications for the treatment of human pathologies.

SMC1A epilepsy syndrome: clinical data from a large international cohort.

SMC1A epilepsy syndrome or developmental and epileptic encephalopathy-85 with or without midline brain defects (DEE85, OMIM #301044) is an X-linked neurologic disorder associated with mutations of the SMC1A gene, which is also responsible for about 5% of patients affected by Cornelia de Lange syndrome spectrum (CdLS). Only described in female patients, SMC1A epilepsy syndrome is characterized by the onset of severe refractory epileptic seizures in the first year of life, global developmental delay, a variable degree of intellectual disability, and dysmorphic facial features not typical of CdLS. This was a descriptive observational study for the largest international cohort with this specific disorder. The main goal of this study was to improve the knowledge of the natural history of this phenotype with particular attention to the psychomotor development and the epilepsy data. The analyzed cohort shows normal prenatal growth with the subsequent development of postnatal microcephaly. The incidence of neonatal problems (seizures and respiratory compromise) is considerable (51.4%). There is a significant prevalence of central nervous system (20%) and cardiovascular malformations (20%). Motor skills are generally delayed. The presence of drug-resistant epilepsy is confirmed; the therapeutic role of a ketogenic diet is still uncertain. The significant regression of previously acquired skills following the onset of seizures has been observed. Facial dysmorphisms are variable and no patient shows a classic CdLS phenotype. To sum up, SMC1A variants caused drug-resistant epilepsy in these patients, more than two-thirds of whom were shown to progress to developmental and epileptic encephalopathy. The SMC1A gene variants are all different from each other (apart from a couple of monozygotic twins), demonstrating the absence of a mutational hotspot in the SMC1A gene. Owing to the absence of phenotypic specificity, whole-exome sequencing is currently the diagnostic gold standard.

CD371-positive pediatric B-cell acute lymphoblastic leukemia: propensity to lineage switch and slow early response to treatment.

ABSTRACT: In the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-Münster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371pos) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371pos. This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371pos BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371pos. In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371pos is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.

Healthcare costs and resources utilization in children with difficult-to-control asthma treated with biologic therapies: A population-based cohort study.

INTRODUCTION: Asthma is one of the most common diseases in children, with a variable range of severity. In recent years, treatment for severe asthma has been largely improved by the availability of targeted biologic therapies. Nevertheless, studies reporting real-world data and cost-effectiveness analyses are lacking. The aim of this study was to evaluate, on a population-based cohort of children with asthma, the impact of the treatment with biologics on healthcare service utilization and associated costs. METHODS: Data were retrieved from Healthcare Utilization database of Lombardy region (Italy). A cohort of 46 asthmatic children aged 6-11 in treatment with dupilumab, mepolizumab or omalizumab was identified during 2017-2021. We compared healthcare resources use between the year before ("baseline period") and the year after the treatment initiation ("follow-up period"). Average 1-year healthcare costs were also calculated. RESULTS: Comparing the baseline with the follow-up period, the number of patients with at least one exacerbation-related hospitalization and ER access decreased by 75.0% and 85.7%, respectively. The use of biologic agents, namely omalizumab, mepolizumab and dupilumab, significantly reduced oral corticosteroids (OCS), short-acting ß2-agonists and the association inhaled corticosteroids/long-acting ß2-agonists use. ER admissions for non-respiratory causes were also significantly reduced, while discontinuation rate was low (6.5%). The overall costs increased, due to the costs of the biologic agents, but the hospital admission-related costs due to respiratory causes reduced significantly. CONCLUSIONS: Our real-world investigation suggests that biologic agents reduced hospital admissions for respiratory causes and use of anti-asthmatic drugs, including OCS. However, long-term healthcare sustainability still needs more in-depth assessments.

Four Additional Doses of PEG-L-Asparaginase During the Consolidation Phase in the AIEOP-BFM ALL 2009 Protocol Do Not Improve Outcome and Increase Toxicity in High-Risk ALL: Results of a Randomized Study.

PURPOSE: The AIEOP-BFM ALL 2009 protocol included, at the end of the induction phase, a randomized study of patients with high-risk (HR) ALL to investigate if an intensive exposure to pegylated L-asparaginase (PEG-ASNASE, 2,500 IU/sqm once a week × 4) on top of BFM consolidation phase IB allowed us to decrease minimal residual disease (MRD) and improve outcome. PATIENTS AND METHODS: A total of 1,097 patients presented, from June 2010 to February 2017, with one or more of the following HR criteria: KMT2A::AFF1 rearrangement, hypodiploidy, prednisone poor response, poor bone marrow response at day 15 (Flow MRD ≥10%), or no complete remission (CR) at the end of induction. Of them, 809 (85.1%) were randomly assigned to receive (404) or not receive (405) four weekly doses of PEG-ASNASE. RESULTS: By intention to treat (ITT) analysis, there was no significant difference in the proportion of patients with polimerase chain reaction MRD ≥5 × 10-4 at the end of phase IB in the experimental versus control arm (13.9% v 17.0%, P = .25). The 5-year event-free survival (median follow-up 6.3 years) by ITT in the experimental and control arms was 70.4% (2.3) versus 75.0% (2.2; P = .18), and the 5-year overall survival was 81.5% (2.0) versus 84.0% (1.9; P = .25), respectively. The corresponding 5-year cumulative incidence of death in CR was 9.5% (1.5) versus 5.7% (1.2; P = .08), and that of relapse was 17.7% (1.9) versus 17.2% (1.9), respectively (P = .94). Adverse reactions in phase IB occurred in 22.2% and 8.9% of patients in the experimental and control arm, respectively (P < .001). CONCLUSION: Additional PEG-ASNASE in phase IB did not translate into a benefit for decreasing relapse incidence but was associated with higher toxicity. Further improvements with conventional chemotherapy might be difficult in the context of intensive treatment protocols.

Application of the Face2Gene tool in an Italian dysmorphological pediatric clinic: Retrospective validation and future perspectives.

Neurodevelopmental disorders exhibit recurrent facial features that can suggest the genetic diagnosis at a glance, but recognizing subtle dysmorphisms is a specialized skill that requires very long training. Face2Gene (FDNA Inc) is an innovative computer-aided phenotyping tool that analyses patient's portraits and suggests 30 candidate syndromes with similar morphology in a prioritized list. We hypothesized that the software could support even expert physicians in the diagnostic workup of genetic conditions. In this study, we assessed the performance of Face2Gene in an Italian dysmorphological pediatrics clinic. We uploaded two-dimensional face pictures of 145 children affected by genetic conditions with typical phenotypic traits. All diagnoses were previously confirmed by cytogenetic or molecular tests. Overall, the software's differential included the correct syndrome in most cases (98%). We evaluated the efficiency of the algorithm even considering the rareness of the genetic conditions. All "common" diagnoses were correctly identified, most of them with high diagnostic accuracy (93% in top-3 matches). Finally, the performance for the most common pediatric syndromes was calculated. Face2Gene performed well even for ultra-rare genetic conditions (75% within top-3 matches and 83% within top-10 matches). Expert geneticists maybe do not need computer support to recognize common syndromes, but our results prove that the tool can be useful not only for general pediatricians but also in dysmorphological clinics for ultra-rare genetic conditions.

A novel flow-cytometric based method to assess post-HSCT donor chimerism exploiting RNA hybridization.

Analysis of donor-recipient chimerism after hematopoietic stem cell transplantation (HSCT) is of pivotal importance for patient's clinical management, especially in the context of mixed chimerism. Patients are routinely monitored for chimerism in sorted subsets of peripheral blood cells. However, measurement of chimerism in sorted immune cell subsets is technically challenging and time consuming. We here propose a novel, flow cytometry-based approach to detect donor cell chimerism in sex-mismatched HSCT. We exploit RNA PrimeFlow™ system, based on RNA hybridization, to detect mRNA from a lysine demethylase encoded by Y chromosome, KDM5D. This approach allows to distinguish male and female derived cells with around 1% sensitivity. The procedure can be coupled with multiparametric immunophenotyping to assess chimerism in specific immune cell subsets without the need for prior FACS-sorting. We apply this method to a cohort of HSCT patients (n = 10) and we show that it is consistent with standard PCR-based method. We also show that different T lymphocyte subsets display variable degrees of donor chimerism, especially in CD8+ T cell compartment where we observe an enrichment for recipient chimerism in central memory T cells. This method can be exploited to advance current knowledge on immune reconstitution focusing on specific subsets avoiding prior FACS-sorting.

Eosinophilic esophagitis: does age matter?

INTRODUCTION: Eosinophilic esophagitis (EoE) is a chronic inflammatory disease of the esophagus with increasing prevalence worldwide. It is a multifactorial disease caused by a combination of immunologic, genetic, and environmental factors. The clinical presentation of EoE varies largely, especially between different age groups. While diagnostic criteria and therapeutic goals are similar in children and adults, there are differences in treatment, with a more cautious approach in children to avoid growth disturbances. In addition, close monitoring and follow-up are essential in children to ensure uninterrupted growth. AREAS COVERED: A search in PubMed/MEDLINE, EMBASE, and SCOPUS databases was conducted to identify relevant studies published between January 2010 and January 2023 to give an overview of the state-of-the-art of EoE epidemiology, diagnosis, and treatment while focusing on similarities and differences between the adult and the pediatric population. EXPERT OPINION: The current state of research indicates that while significant progress has been made in understanding and treating EoE, further research and advances are needed to optimize diagnostic strategies, tailored treatment approaches, monitoring, and follow-up, and improve long-term outcomes for patients. With further innovation, the management of EoE can become more precise and tailored, leading to better patient outcomes and improved quality of life.

Functional relevance of circRNA aberrant expression in pediatric acute leukemia with KMT2A::AFF1 fusion.

ABSTRACT: Circular RNAs (circRNAs) are emerging molecular players in leukemogenesis and promising therapeutic targets. In KMT2A::AFF1 (MLL::AF4)-rearranged leukemia, an aggressive disease compared with other pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), data about circRNAs are limited. Here, we disclose the circRNA landscape of infant patients with KMT2A::AFF1 translocated BCP-ALL showing dysregulated, mostly ectopically expressed, circRNAs in leukemia cells. Most of these circRNAs, apart from circHIPK3 and circZNF609, previously associated with oncogenic behavior in ALL, are still uncharacterized. An in vitro loss-of-function screening identified an oncogenic role of circFKBP5, circKLHL2, circNR3C1, and circPAN3 in KMT2A::AFF1 ALL, whose silencing affected cell proliferation and apoptosis. Further study in an extended cohort disclosed a significantly correlated expression of these oncogenic circRNAs and their putative involvement in common regulatory networks. Moreover, it showed that circAFF1 upregulation occurs in a subset of cases with HOXA KMT2A::AFF1 ALL. Collectively, functional analyses and patient data reveal oncogenic circRNA upregulation as a relevant mechanism that sustains the malignant cell phenotype in KMT2A::AFF1 ALL.

Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy has shown unprecedented results in patients with B cell relapsed/refractory acute lymphoblastic leukemia (R/R-ALL) and B cell non-Hodgkin lymphomas where no other curative options are available. In vivo monitoring of CAR-T cell kinetics is fundamental to understand the correlation between CAR-T cells expansion and persistence with treatment response and toxicity development. The aim of this study was to define a robust, sensitive, and universal method for CAR-T cell detection using flow cytometry. We set up and compared with each other three assays for CD19 CAR-T cell detection, all based on commercially available reagents. All methods used a recombinant human CD19 protein fragment recognized by the single-chain variable fragment of the CAR construct. The two indirect staining assays (CD19his + APC-conjugated antihistidine antibody and CD19bio + APC-conjugated antibiotin antibody) showed better sensitivity and specificity compared with the direct staining with CD19-FITC, and CD19his had a better cost-effective profile. We validated CAR detection with CD19his with parallel quantitative real-time polymerase chain reaction data and we could demonstrate a strong positive correlation. We also showed that CD19his staining can be easily included in a multicolor flow cytometry panel to achieve additional information about the cell phenotype of CAR-T cell positive subpopulations. Finally, this method can be used for different anti-CD19 CAR-T cell products and for different sample sources. These data demonstrate that detection of CAR-T cells by CD19his flow cytometry staining is a reliable, robust, and broadly applicable tool for in vivo monitoring of CAR-T cells.

Phenotypic profiling of CD34+ cells by advanced flow cytometry improves diagnosis of juvenile myelomonocytic leukemia.

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B-cell and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single eight-color antibody combination.