Corrigendum: Impact of disease burden and late loss of B cell aplasia on the risk of relapse after CD19 chimeric antigen receptor T Cell (Tisagenlecleucel) infusion in pediatric and young adult patients with relapse/refractory acute lymphoblastic leukemia: role of B-cell monitoring.

[This corrects the article DOI: 10.3389/fimmu.2023.1280580.].

The shape of cancer relapse: Topological data analysis predicts recurrence in paediatric acute lymphoblastic leukaemia.

Although children and adolescents with acute lymphoblastic leukaemia (ALL) have high survival rates, approximately 15-20% of patients relapse. Risk of relapse is routinely estimated at diagnosis by biological factors, including flow cytometry data. This high-dimensional data is typically manually assessed by projecting it onto a subset of biomarkers. Cell density and "empty spaces" in 2D projections of the data, i.e. regions devoid of cells, are then used for qualitative assessment. Here, we use topological data analysis (TDA), which quantifies shapes, including empty spaces, in data, to analyse pre-treatment ALL datasets with known patient outcomes. We combine these fully unsupervised analyses with Machine Learning (ML) to identify significant shape characteristics and demonstrate that they accurately predict risk of relapse, particularly for patients previously classified as 'low risk'. We independently confirm the predictive power of CD10, CD20, CD38, and CD45 as biomarkers for ALL diagnosis. Based on our analyses, we propose three increasingly detailed prognostic pipelines for analysing flow cytometry data from ALL patients depending on technical and technological availability: 1. Visual inspection of specific biological features in biparametric projections of the data; 2. Computation of quantitative topological descriptors of such projections; 3. A combined analysis, using TDA and ML, in the four-parameter space defined by CD10, CD20, CD38 and CD45. Our analyses readily extend to other haematological malignancies.

Monitoring of kinetics and exhaustion markers of circulating CAR-T cells as early predictive factors in patients with B-cell malignancies.

Purpose: CAR-T cell therapy has proven to be a disruptive treatment in the hematology field, however, less than 50% of patients maintain long-term response and early predictors of outcome are still inconsistently defined. Here, we aimed to optimize the detection of CD19 CAR-T cells in blood and to identify phenotypic features as early biomarkers associated with toxicity and outcomes. Experimental design: In this study, monitoring by flow cytometry and digital PCR (dPCR), and immunophenotypic characterization of circulating CAR-T cells from 48 patients treated with Tisa-cel or Axi-cel was performed. Results: Validation of the flow cytometry reagent for the detection of CAR-T cells in blood revealed CD19 protein conjugated with streptavidin as the optimal detection method. Kinetics of CAR-T cell expansion in blood confirmed median day of peak expansion at seven days post-infusion by both flow cytometry and digital PCR. Circulating CAR-T cells showed an activated, proliferative, and exhausted phenotype at the time of peak expansion. Patients with increased expansion showed more severe CRS and ICANs. Immunophenotypic characterization of CAR-T cells at the peak expansion identified the increased expression of co-inhibitory molecules PD1 and LAG3 and reduced levels of the cytotoxicity marker CD107a as predictors of a better long-term disease control. Conclusions: These data show the importance of CAR-T cells in vivo monitoring and identify the expression of PD1LAG3 and CD107a as early biomarkers of long-term disease control after CAR-T cell therapy.

Impact of disease burden and late loss of B cell aplasia on the risk of relapse after CD19 chimeric antigen receptor T Cell (Tisagenlecleucel) infusion in pediatric and young adult patients with relapse/refractory acute lymphoblastic leukemia: role of B-cell monitoring.

Introduction: Loss of B-cell aplasia (BCA) is a well-known marker of functional loss of CD19 CAR-T. Most relapses and loss of BCA occur in the first months after CD19 CAR-T infusion. In addition, high tumor burden (HTB) has shown to have a strong impact on relapse, especially in CD19-negative. However, little is known about the impact of late loss of BCA or the relationship between BCA and pre-infusion tumor burden in patients infused with tisagenlecleucel for relapsed/refractory B-cell acute lymphoblastic leukemia. Therefore, the optimal management of patients with loss of BCA is yet to be defined. Methods: We conducted a Spanish, multicentre, retrospective study in patients infused with tisagenlecleucel after marketing authorization. A total of 73 consecutively treated patients were evaluated. Results: Prior to infusion, 39 patients had HTB (≥ 5% bone marrow blasts) whereas 34 had a low tumor burden (LTB) (<5% blasts). Complete remission was achieved in 90.4% of patients, of whom 59% relapsed. HTB was associated with inferior outcomes, with a 12-month EFS of 19.3% compared to 67.2% in patients with LTB (p<0.001) with a median follow-up of 13.5 months (95% CI 12.4 - 16.2). In the HTB subgroup relapses were mainly CD19-negative (72%) whereas in the LTB subgroup they were mainly CD19-positive (71%) (p=0.017). In the LTB group, all CD19-positive relapses were preceded by loss of BCA whereas only 57% (4/7) of HTB patients experienced CD19-positive relapse. We found a positive correlation between loss of BCA and CD19-positive relapse (R-squared: 74) which persisted beyond six months post-infusion. We also explored B-cell recovery over time using two different definitions of loss of BCA and found a few discrepancies. Interestingly, transient immature B-cell recovery followed by BCA was observed in two pediatric patients. In conclusion, HTB has an unfavorable impact on EFS and allo-SCT might be considered in all patients with HTB, regardless of BCA. In patients with LTB, loss of BCA preceded all CD19-positive relapses. CD19-positive relapse was also frequent in patients who lost BCA beyond six months post-infusion. Therefore, these patients are still at significant risk for relapse and close MRD monitoring and/or therapeutic interventions should be considered.

High-Dimensional Analysis of Single-Cell Flow Cytometry Data Predicts Relapse in Childhood Acute Lymphoblastic Leukaemia.

Artificial intelligence methods may help in unveiling information that is hidden in high-dimensional oncological data. Flow cytometry studies of haematological malignancies provide quantitative data with the potential to be used for the construction of response biomarkers. Many computational methods from the bioinformatics toolbox can be applied to these data, but they have not been exploited in their full potential in leukaemias, specifically for the case of childhood B-cell Acute Lymphoblastic Leukaemia. In this paper, we analysed flow cytometry data that were obtained at diagnosis from 56 paediatric B-cell Acute Lymphoblastic Leukaemia patients from two local institutions. Our aim was to assess the prognostic potential of immunophenotypical marker expression intensity. We constructed classifiers that are based on the Fisher's Ratio to quantify differences between patients with relapsing and non-relapsing disease. We also correlated this with genetic information. The main result that arises from the data was the association between subexpression of marker CD38 and the probability of relapse.

Blinatumomab and inotuzumab for B cell precursor acute lymphoblastic leukaemia in children: a retrospective study from the Leukemia Working Group of the Spanish Society of Pediatric Hematology and Oncology (SEHOP).

Blinatumomab and inotuzumab ozogamycin represent promising alternatives to conventional chemotherapy in acute lymphoblastic leukaemia (ALL). We analysed data from 29 children with ALL treated under compassionate use with blinatumomab, inotuzumab or both. The complete remission (CR) rate in a heavily pretreated population with overt relapse was 47·6%. At earlier stages (first/second CR), both antibodies represented a useful tool to reduce minimal residual disease, and/or avoid further toxic chemotherapy until stem cell transplantation. Six patients developed grade 3 reversible non-haematological toxicity. The 12-month overall survival and event-free survival rates were 50·8 ± 26·4% and 38·9 ± 25·3% with blinatumomab, 45·8 ± 26% and 27·5 ± 25% with inotuzumab.

Measurable Residual Disease Assessed by Flow-Cytometry Is a Stable Prognostic Factor for Pediatric T-Cell Acute Lymphoblastic Leukemia in Consecutive SEHOP Protocols Whereas the Impact of Oncogenetics Depends on Treatment.

Robust and applicable risk-stratifying genetic factors at diagnosis in pediatric T-cell acute lymphoblastic leukemia (T-ALL) are still lacking, and most protocols rely on measurable residual disease (MRD) assessment. In our study, we aimed to analyze the impact of NOTCH1, FBXW7, PTEN, and RAS mutations, the measurable residual disease (MRD) levels assessed by flow cytometry (FCM-MRD) and other reported risk factors in a Spanish cohort of pediatric T-ALL patients. We included 199 patients treated with SEHOP and PETHEMA consecutive protocols from 1998 to 2019. We observed a better outcome of patients included in the newest SEHOP-PETHEMA-2013 protocol compared to the previous SHOP-2005 cohort. FCM-MRD significantly predicted outcome in both protocols, but the impact at early and late time points differed between protocols. The impact of FCM-MRD at late time points was more evident in SEHOP-PETHEMA 2013, whereas in SHOP-2005 FCM-MRD was predictive of outcome at early time points. Genetics impact was different in SHOP-2005 and SEHOP-PETHEMA-2013 cohorts: NOTCH1 mutations impacted on overall survival only in the SEHOP-PETHEMA-2013 cohort, whereas homozygous deletions of CDKN2A/B had a significantly higher CIR in SHOP-2005 patients. We applied the clinical classification combining oncogenetics, WBC count and MRD levels at the end of induction as previously reported by the FRALLE group. Using this score, we identified different subgroups of patients with statistically different outcome in both Spanish cohorts. In SHOP-2005, the FRALLE classifier identified a subgroup of high-risk patients with poorer survival. In the newest protocol SEHOP-PETHEMA-2013, a very low-risk group of patients with excellent outcome and no relapses was detected, with borderline significance. Overall, FCM-MRD, WBC count and oncogenetics may refine the risk-stratification, helping to design tailored approaches for pediatric T-ALL patients.

Absence of WASp Enhances Hematopoietic and Megakaryocytic Differentiation in a Human Embryonic Stem Cell Model.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.

Emerging BCR/ABL1 Mutations Under Treatment with Tyrosine Kinase Inhibitors in Paediatric Acute Lymphoblastic Leukaemia.

We report on the emergence and clinical relevance of an unusual BCR-ABL1 kinase domain mutational status in a 2-year-old female with p210-BCR-ABL Philadelphia chromosome-positive acute lymphoblastic leukaemia. We detected three BCR-ABL1 clones determined by the presence of the E255V, D276G and F317L mutations. We point out the usefulness of searching for mutated populations that survive tyrosine-kinase inhibitor therapy and the role of their clonal selection over time in relation to therapeutic intervention.

Epidemiología de la colonización nasofaríngea por Streptococcus pneumoniae en niños menores de 6 años de la ciudad de Sevilla / Epidemiology of nasopharyngeal carriage of Streptococcus pneumoniae in children < 6 years old in Seville

Introducción: El objetivo de la investigación fue estudiar la epidemiología de la colonización nasofaríngea por Streptococcus pneumoniae (S. pneumoniae) tras la introducción de la vacuna conjugada neumocócica heptavalente (VCN7).Métodos Se obtuvieron muestras NF en 848 niños > 6 meses y<6 años atendidos en cuatro centros de salud (niños sanos) y en dos urgencias hospitalarias (niños enfermos) de Sevilla durante el período comprendido entre el 1/2/2005 y el 31/6/2008. Resultados De forma global 278 (33%) niños estaban colonizados por S. pneumoniae, la asistencia a guardería o colegio (OR: 2,21; IC 95%: 1,54-3,15; p=0,0001) y la edad < 3 años (OR: 1,87; IC 95%: 1,3-2,69; p=0,001) fueron predictores independientes de la colonización neumocócica. La utilización reciente de antibióticos tuvo un efecto protector (OR: 0,68; IC 95%: 0,48-0,94; p=0,02). Cobertura vacunal VCN7 41%. Se encontró una menor frecuencia de colonización por serotipos vacunales (SV) en aquellos niños (..) (AU)

Introduction: The aim of this investigation was to study the epidemiology of nasopharyngeal (NP) colonization with Streptococcus pneumoniae after the introduction of the heptavalent pneumococcal conjugate vaccine (PCV7).Methods: NP swabs were obtained from 848 children aged 6 months to six years seen in four primary care centres (healthy children) and in two emergency depeartments (sick children) from Seville. The study was conducted between February 2005 and June 2008.Results: A total of 278 (33%) children carried S. pneumoniae. Pneumococcal colonization was independently predicted by school attendance or child care participation (OR 2.21; 95% CI 1.54- 3.15; P=.0001)and younger age. Recent antibiotic use was protective (OR 0.68; 95% CI 0.48-0.94; P=.02). PCV7 uptake was 41%. Risk of carriage of PCV7- type pneumococci was lower among children who had (..) (AU)

[Epidemiology of nasopharyngeal carriage of Streptococcus pneumoniae in children < 6 years old in Seville]. / Epidemiología de la colonización nasofaríngea por Streptococcus pneumoniae en niños menores de 6 años de la ciudad de Sevilla.

INTRODUCTION: The aim of this investigation was to study the epidemiology of nasopharyngeal (NP) colonization with Streptococcus pneumoniae after the introduction of the heptavalent pneumococcal conjugate vaccine (PCV7). METHODS: NP swabs were obtained from 848 children aged 6 months to six years seen in four primary care centres (healthy children) and in two emergency depeartments (sick children) from Seville. The study was conducted between February 2005 and June 2008. RESULTS: A total of 278 (33%) children carried S. pneumoniae. Pneumococcal colonization was independently predicted by school attendance or child care participation (OR 2.21; 95% CI 1.54- 3.15; P=.0001) and younger age. Recent antibiotic use was protective (OR 0.68; 95% CI 0.48-0.94; P=.02). PCV7 uptake was 41%. Risk of carriage of PCV7- type pneumococci was lower among children who had received ≥1 dose of PCV7 (7% vs 29%; [OR 0.21; 95% CI 0.09-0.49; P=.0001]). The proportion of pneumococcal isolates with oral penicillin non-susceptibility and amoxicillin resistance were 33% and 3%, respectively. Amoxicillin resistance in colonized children was associated with prior antibiotic usage (OR 4.29; 95% CI 1.09-20.02). CONCLUSIONS: NP colonization rates with PCV7- type pneumococci were low compared to those found in studies prior to PCV7 introduction, both in vaccinated and unvaccinated subjects. Factors related to age and overcrowding increased the prevalence of pneumococcal carriage. Use of antibiotics reduced the overall carriage of pneumococci, but was a risk factor for colonization with amoxicillin resistant pneumococci.

Pyogenic sacroiliitis in children-a diagnostic challenge.

Osteoarticular infections in paediatric patients are associated with significant morbidity. Pyogenic sacroiliitis is rare and accounts for approximately 1-2% of osteoarticular infections in children. Diagnosis of this disease has been difficult in the past due to its deep location and may be delayed due to the lack of specific clinical signs and symptoms. We identified 11 paediatric patients with clinical-radiological signs of pyogenic sacroiliitis during an 8-year period and observed an unusual cluster of four cases during the last 11 months. Early diagnosis was possible due to a reproducible clinical pattern as well as radiological evidences of infection using magnetic resonance imaging and/or bone scintigraphy; most patients having predisposing factors. Staphylococcus aureus was the sole causative agent identified. All patients including two children with associated muscle abscesses were managed conservatively with antibiotic therapy only and showed good clinical response with no sequelae during follow-up. An algorithm for the correct and prompt diagnosis of this pathology is proposed. Standardised optimal therapy remains to be defined.