Pediatric Acute Lymphoblastic Leukemia Patients Research Articles

Hydrocortisone or Placebo to Reduce Dexamethasone-Induced Neurobehavioral Side Effects: Results of a Phase 3 National, Double-Blind, Placebo-Controlled Randomized Trial with Crossover Design

Background The introduction of dexamethasone for the treatment of pediatric acute lymphoblastic leukemia (ALL) significantly contributed to the current overall survival rate of more than 90% in high-income countries. The downside however, is that dexamethasone can induce serious side effects, of which neurobehavioral side effects are experienced as detrimental with respect to health-related quality of life (HRQoL) by both patients and parents. Our previous study suggests that children who suffer most from neurobehavioral side effects might benefit from physiological hydrocortisone addition to dexamethasone treatment. We aimed to validate this finding in a prospective large cohort of pediatric ALL patients. Methods Our phase 3, double-blind, randomized controlled trial with crossover design included ALL patients (3-18 years) during medium risk maintenance therapy in a national tertiary hospital between May 17, 2018 and August 5, 2020. A baseline measurement before and after one five-day dexamethasone course was performed to identify patients with clinically relevant dexamethasone-induced neurobehavioral problems, defined as a rise of ≥5 points on the Strengths and Difficulties Questionnaire (SDQ) Total Difficulties score. These patients were subsequently randomized to receive an intervention during the following four five-day dexamethasone courses. The intervention consisted of two courses hydrocortisone (physiological dose of 10mg/m2/day in a circadian rhythm), followed by two courses placebo, or vice versa. Neurobehavioral problems were assessed before and after each course using the parent-reported SDQ as primary endpoint. Secondary endpoints were sleep problems, HRQoL, hunger feeling, and parental stress, measured with separate questionnaires and actigraphy. A generalized mixed model was estimated to study the intervention effect. Findings Of 79 included patients, 52 (66%) experienced clinically relevant dexamethasone-induced neurobehavioral problems. Both hydrocortisone and placebo significantly and relevantly decreased dexamethasone-induced neurobehavioral problems measured with the SDQ (estimated effect hydrocortisone: -8.19 (95%-confidence interval (CI) -12.35 to -4.04), estimated effect placebo: -6.75 (95%-CI -10.95 to -2.56) (table 1 and figure 1). This indicates that, when adding hydrocortisone or placebo to dexamethasone treatment, the SDQ Total Difficulties score is estimated to be 8.19 or 6.75 points lower than without the addition of hydrocortisone or placebo. Since one SD of SDQ norm values is between 4 and 6 points, the decrease on the SDQ after hydrocortisone or placebo addition is more than 1 SD, and therefore clinically significant. The strength of the effect was similar in both interventions. There was no benefit from hydrocortisone or placebo in reducing sleep problems, hunger, parental stress or improving HRQoL. Interpretation Clinically relevant dexamethasone-induced neurobehavioral problems decreased equally and significantly after both hydrocortisone and placebo addition. Using the placebo effect in clinical practice could be a powerful and innovative way to diminish neurobehavioral problems in children who experience clinically relevant dexamethasone-induced side effects. Moreover, the high proportion of patients who experience clinically relevant dexamethasone-induced neurobehavioral problems might be caused by a nocebo effect of dexamethasone. By offering both interventions (hydrocortisone and open-label placebo) in clinical practice and by diminishing the nocebo effect of dexamethasone, we hope to improve dexamethasone-induced side effects in those patients who suffer most. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mutational Mechanisms in Pediatric Acute Lymphoblastic Leukemia with Multiple Relapses

Pediatric acute lymphoblastic leukemia (ALL) is generally marked by a low mutational load at initial diagnosis, which increases in subsequent relapses, partly because of hypermutation in a subset of cases (Waanders et al., 2020, Blood Canc. Disc., 1:96). This increased mutational load can be driven by intrinsic mutational processes or could be therapy related. In this study, we have investigated these mutational processes in pediatric ALL patients who suffered multiple relapses, to determine which processes are active in (relapsed) ALL and how they behave during disease progression before and after therapy. We identified 63 historical cases of ALL with multiple relapses, and selected patients by availability of material and blast percentage above 70% in multiple leukemic samples. If viable material with less than 70% blasts was available, mononuclear cells were enriched by sorting for CD10 positivity. We collected and whole genome sequenced 97 tumor samples from 28 ALL patients (27 precursor B-ALL and 1 T-ALL) with 2 (n=17) or more (n=11) relapses, and 44 remission samples. Somatic single nucleotide variants (SNVs) were called by Mutect2. SNVs were clustered per sample based on their dynamics in time and all clusters in all patients were included for de novo signature extraction using the R package MutationalPatterns. A mutational process was deemed active in a sample if >150 SNVs and >10% of SNVs in a cluster were attributed to a mutational process by de novo signature extraction. The majority of ALL patients with multiple relapses had a mutational load above average, compared to an unselected diagnosis cohort, which often increased during disease progression. Six cases (21%) passed the hypermutation threshold of 1.3 mutations/Mb as previously defined (Waanders et al., 2020). However, mutational signature analysis revealed that mutational processes were active in a much larger proportion of cases. We extracted 7 single base substitution (SBS) signatures, of which 4 were known COSMIC signatures (cosine similarity >0.95) and 2 were similar to known COSMIC signatures (cosine similarity >0.8). At time of diagnosis 4 distinct mutational processes were found to be active alone or in combination in 10 cases (36%), which is much higher than an unselected diagnosis cohort (13%). These processes included APOBEC-associated mutagenesis (SBS2 & SBS13), a mutational pattern resembling UV-like damage (SBS7a), a process that may be caused by reactive oxygen species (SBS18-like) and an unknown process (Fig. 1). By clustering SNVs per patient in time we followed the activity of mutational processes and identified rising and falling (sub)clones. APOBEC mutagenesis was found to be an ongoing process, sometimes appearing for the first time at relapse (Fig. 1B), although when xenografted in mice (n=2) no continued mutagenesis was observed. SBS7a or SBS18-like mutagenesis always occurred already at diagnosis. One patient, who relapsed 5 times, acquired at each presentation between 500-1500 new SNVs with a specific pattern that did not match any known cosmic mutational signatures, and may represent a novel intrinsic mutational mechanism. At time of relapse, additional (mostly treatment-related) mutational processes became active, eventually affecting 17/28 (61%) patients (Fig. 1A), much higher than observed in cases with single relapses (~40%; Li et al., 2020, Blood, 135:41). We observed the thiopurine-associated SBS87 in 9 (32%) patients, and an unexplained SBS86-like mutational signature in 2 patients (Fig. 1B). Furthermore, we noticed SBS17-like mutagenesis in one patient. The etiology of SBS17 is as yet not established, but it has been proposed to be caused by reactive oxygen species. To conclude, ALL patients with multiple relapses showed an increased prevalence and large variety of interplaying mutational processes at diagnosis (36%) and final relapse (61%), with intrinsic or therapy induced origins. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Hemostatic Profile at Diagnosis in Childhood Acute Lymphoblastic Leukemia: A Meta-Analysis of Cases from the Literature

Introduction Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer with, an incidence rate of 7.55 cases per 100,000 person-years for males aged 0-4 in the Canadian population. Thromboembolism (TE) is a well-recognized serious complication associated with ALL that results in significant morbidity and occasionally mortality. In patients with ALL, the reported incidence of symptomatic and asymptomatic TE is as high as 16% and 37%, respectively. At diagnosis, there is evidence of increased thrombin generation in children with ALL. Patients often present with a hypercoagulable state and this prothrombotic condition persists during the treatment period, indicating a possible interaction of the disease and therapy. Since the efficacy and safety of thromboprophylaxis during ALL treatment are still being explored, understanding the biology of the disease is crucial for defining the optimal strategy for prevention and management of ALL-associated thrombosis. In this study, we aim to explore a better understanding of how ALL affects the hemostatic balance. Due to the low incidence of leukemia, single-center studies are not adequately powered to detect significance. This meta-analysis was conducted to comprehensively evaluate the hemostatic profile at the time of diagnosis in pediatric ALL patients. Method PubMed database was searched on December 2021 with the following keywords: "acute lymphoblastic leukemia", "coagulation", "fibrinolysis" and "hemostatic." The inclusion criteria were: 1) study participants with childhood ALL, 2) hemostatic parameters were measured and reported at the time of diagnosis, 3) there is a comparator group of healthy subjects, and 4) there is no other reported comorbidities. Non-human studies, non-English articles and studies without healthy comparators were excluded. Data on coagulation profile at diagnosis were collected and meta-analyzed. The statistical method was random-effect inverse-variance weighting. The results were reported as mean difference with 95% confidence intervals (CIs) Result The initial search yielded 643 articles. Of these, 505 studies were excluded from title/abstract screening. The fulltexts of the remaining studies were screened, and 33 studies were included for the final analysis. D-dimer levels were elevated in the Leukemic group by 533.73 ng/ml [95% CIs: 283.1-782.35], p<0.01, n=216,193. Thrombin-antithrombin complex levels were also increased in the Leukemic group by 109.89 ug/L [95% CIs: 77.89-141.89], p<0.01, n=149,136. In addition, the levels of natural anticoagulants were reduced in the Leukemic group. Protein C activities were lower in the Leukemic group by 20.29% [95% CIs: 2.14-38.44], p<0.01, n=146,115. Protein S activities were lower in the Leukemic group by 29.11% [95% CIs: -1.28-59.5], p<0.01, n=146,115. Furthermore, markers of endothelial dysfunction were elevated in the Leukemic group. VWF levels were increased by 28.26% [95% CIs: -1.64-58.17], p<0.01, n=133,205 while tPA levels were reduced by 631.15 pg/ml [95% CIs: 296.22-966.09], p<0.01, n=122,80. Significance Children with ALL already present with hemostatic imbalance at diagnosis, favoring a hypercoagulable state. Interestingly, there has been little research on thrombin markers prior to chemotherapy in childhood ALL. The majority of data were extracted from prospective studies investigating the effect of therapy on thrombosis. Our study highlights significant differences from healthy control levels in D-dimer, Thrombin-antithrombin complex, protein C activity, protein S activity, VWF and tPA levels in children with ALL at diagnosis. Better tools of prediction would allow prospective identification of patients in whom prophylaxis might reduce the risk of thrombosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pediatric Acute Lymphoblastic Leukemia with Pdgfrb Fusions: A Multicenter Retrospective Study

Objective: PDGFRB fusions in acute lymphoblastic leukemia (ALL) is rare. PDGFRB fusions was mainly observed in B-ALL, but also in T-ALL. Previous studies were mostly limited to anecdotal cases, suggesting that this subtype of ALL had low rate of remission and poor prognosis. The addition of tyrosine kinase inhibitor (TKI) to intensive chemotherapy may improve the outcome, but lack of long-term survival data. Herein, we retrospectively analyzed the clinical characteristics, survival and prognostic factors for 28 pediatric patients with PDGFRB fusions ALL. Methods: This multicenter, retrospective study included pediatric patients with newly diagnosed PDGFRB fusion ALL after treatments according to the CCCG-ALL-2015 and CCCG-ALL-2020 protocols from April 2015 to April 2022 in 10 hospitals in China. All patients completed the followed up. The baseline characteristics and treatment outcomes of these patients, including complete remission, drug resistance, relapse, treatment-related mortality and minimal residual disease (MRD) at end of induction therapy, as well as hematopoietic stem cell transplantation (HSCT) were collected. The outcome was compared between patients treated with and without TKI. Fisher test and nonparametric rank sum test were used for comparative analysis. 4-year event-free survival (EFS) and 4-year overall survival (OS) were estimated by use of Kaplan-Meier methods, and the 4-year cumulative incidence of relapse was calculated by use of a competing risk model gray test. Univariate and multivariate analyses were performed by use of Cox regression. Results: We identified 28 pediatric patients with newly diagnosed PDGFRB fusion ALL, which accounted for approximately 0.5% of childhood ALL. In all 28 patients, 21 (75%) were male and 7 (25%) were female; the median age was 10 (1-16) years old; median white blood cell (WBC) was 29.8 (1.6-627.7) × 10^9/L; 24 (85.7%) were acute B-lymphoblastic leukemia and 4 (14.3%) were acute T-lymphoblastic leukemia; one case (3.5%) was observed accompanied by the CNS involvement at diagnosis. Twenty of the patients received TKI treatment. Only 6 patients underwent hematopoietic stem cell transplantation. For all 28 patients, the median follow-up was 25.5 (3-77) months, the 4-year EFS was 29.2% (95% CI, 13.6-36.4), the 4-year OS was 42.1% (95% CI, 30.6-61.4), and the 4-year cumulative incidence of relapse was 58% (95% CI, 45·6-70·1). Patients in TKI group had a high rate of complete remission after induction therapy compared with those in no-TKI group (90% vs 63.6%). However, no significant differences were observed in the 4-year EFS and 4-year OS between TKI group and no-TKI group (EFS, 21.1% vs 43.8%, P=0.925; OS, 50.1% vs 36.5%, P=0.807). The cumulative incidences of relapse between these two groups also have no remarkable differences (P >0.05). Among the 6 patients who underwent HSCT, 2 patients who achieved CR1 survived and 4 died. Univariate analysis showed that minimal residual disease (MRD) ≥10-4 after induction therapy for D46 and IKZF1 positive had a poor prognosis (all P <0.05). MRD≥10-4 at the end of induction therapy is an independent risk factor for poor prognosis (hazard ratio of event-free, 9.101 [95% CI 2.03-40.86], P =0·004, compared to those with a MRD of <10-4). Conclusion: Pediatric ALL patients with PDGFRB fusions have a poor outcome. This subtype of ALL is associated with high-risk clinical features such as older age, high WBC count at diagnosis, high MRD after induction therapy, and increased risk of leukemia relapse. The addition of TKI to chemotherapy can improve the early remission rate of treatment, but does not improve the long-term survival. Profitably, the TKI treatment can provide an opportunity for subsequent treatment, including HSCT. Additionally, HSCT has indeed potential to reduce the relapse rate for this subtype patients. HSCT is recommended for this subtype patients who achieve CR1. The MRD≥10-4 or more at the end of induction therapy was independently predictive factor for unfavorable outcome and stratified treatment based on MRD may improve survival of these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Severe Adverse Events Associated with Concentrations of High Dose Methotrexate in Pediatric Acute Lymphoblastic Leukemia Patients

Severe Adverse Events Associated with Concentrations of High Dose Methotrexate in Pediatric Acute Lymphoblastic Leukemia Patients

Tcrαβ+/CD19+-Depletion in Hematopoietic Stem Cells Transplantation from Matched Unrelated and Haploidentical Donors in Pediatric Acute Lymphoblastic Leukemia Patients in Complete Remission

Tcrαβ+/CD19+-Depletion in Hematopoietic Stem Cells Transplantation from Matched Unrelated and Haploidentical Donors in Pediatric Acute Lymphoblastic Leukemia Patients in Complete Remission

Universal Premedication for Pegylated (PEG)-Asparaginase Treatment in Children with Acute Lymphoblastic Leukemia (ALL) and Lymphoblastic Lymphoma (LLy) Has No Impact on Frequency of Hypersensitivity Reactions

Asparaginase is an established and critical chemotherapeutic agent in the treatment of Acute Lymphoblastic Leukemia (ALL) and Lymphoblastic Lymphoma (LLy). Currently, pegylated asparaginase (PEG-Asp), an E. coli derived enzyme, is the first-line asparaginase preparation used in most contemporary ALL therapy protocols. Hypersensitivity reactions are one of the main toxicities of PEG-Asp therapy occurring in up to 30% of patients (1) and is one of most common reasons for discontinuation of PEG-Asp requiring substitution. Infusion reactions are also seen in patients receiving intravenous (IV) PEG-Asp, but are clinically difficult to differentiate from hypersensitivity reactions, which are associated with neutralizing anti-asparaginase antibody-production. Reactions lead to a switch to more costly and more frequent treatment with Erwinia Asparaginase, an immunologically distinct formulation. Pre-medication with H1/H2 blockers has been shown to reduce the incidence of infusion reactions (2). This has led to adoption of premedication with H1/H2 blockers, including at our centre. However, the data remain mixed, with some data supporting and some disputing this claim (3-4). There also remain concerns regarding premedication masking of milder reactions due to neutralizing antibodies and as such we have implemented serum asparaginase activity (SAA) monitoring along with premedication. This study aimed to assess the impact of pre-medication with H2 blockers and diphenhydramine for PEG-Asparaginase treatment as well as to create a cost analysis to determine the cost-savings effects of universal premedication. To achieve this, we performed a retrospective chart review at the McMaster Children's Hospital comparing rates of hypersensitivity reactions in pediatric leukemia and lymphoma patients prior to universal pre-medication (PM-) from 2016-2018 and after implementation (PM+) from 2019-2021 at our centre. We also assessed the cost-saving effects of universal premedication at our centre. We reviewed a total of 107 charts of pediatric patients with ALL & LLy cared for at our centre over the period of 2016 - 2021. Our results demonstrate that there was no significant difference in reactions between PM- and PM+ patients across all risk stratifications (p = 0.25, CI -0.25, +0.07). There was no difference in median CTCAE grade reactions. There was a total of 2 patients that were diagnosed as a silent inactivation utilizing SAA monitoring. These findings suggest that universal premedication had no impact on the rate of hypersensitivity reactions or CTCAE grade of reaction in patients receiving PEG-Asparaginase, but SAA monitoring has proven effective in diagnosing silent inactivation that may have otherwise been missed. In determining the costs of universal pre-medication per patient, the cost at our centre ranged from $10.88 - $85.14 depending on ALL risk stratification as well as weight-based doses. This does not include the costs for administering the IV medications, nursing costs, time added to care, and hospital waste including vials and syringes. There was no statistically significant difference in number of Erwinia doses administered between groups. Our limitations include the retrospective nature of the study and small sample size. In conclusion, our data suggest that universal premedication did not impact the number and grade of hypersensitivity reactions to PEG-Asp, nor the number of Erwinia doses needed to be substituted. Larger studies are recommended to confirm our findings. We recommend continued SAA monitoring for patients receiving PEG-Asparaginase to identify patients with silent inactivation of asparaginase. References 1) Raetz EA, Salzer WL. Tolerability and efficacy of L-asparaginase therapy in pediatric patients with acute lymphoblastic leukemia. J Pediatr Hematol Oncol. 2010 Oct;32(7):554-63 2) Kurtzberg, J., et al (1993). Antibodies to asparaginase alter pharmacokinetics and decrease enzyme activity in patients on asparaginase therapy. In Proc Am Assoc Cancer Res (Vol. 34, p. 304). 3) Menig, S., et al. (2016). The Impact of PEG-Aspargase Premedication on Hypersensitivity Reaction in Pediatric Acute Lymphoblastic Leukemia (ALL) and Lymphoblastic Lymphoma (LLy) Patients. Poster Presentation. 4) Young, D., et al. Universal premedication for asparaginase-based therapy should be standard of care. ASPHO Conference Presentation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dormancy Is a Feature of Therapy Resistant Mouse and Human Leukemia-Initiating Cells in T-ALL

Addressing therapy failure and relapse is critical in improving outcomes for pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Relapse is thought to represent a failure to effectively eliminate leukemia-initiating cells (L-ICs), which survive chemotherapy and self-renew, regenerating the full complement of leukemic cell populations. Results from mouse T-ALL models show that the double negative-3 (DN3) thymic progenitors are enriched for L-IC activity; however, the L-IC remains an undefined subset of this population. We used single cell RNA-sequencing (scRNA-seq) to profile leukemic development in vivo, with a focus on uncovering DN3 heterogeneity. Integrated UMAP clustering of scRNA-seq libraries from wild type (WT), Tal1/Lmo2 preleukemic (PL), and Tal1/Lmo2 T-ALL (Leuk) thymi revealed an expansion of DN3 progenitors in PL and Leuk samples with decreases in mature (single positive) thymocytes. Unlike WT thymocytes, where Notch1 target gene expression was restricted to DN3 cells, Notch1-Myc pathway activation was maintained throughout thymocyte development in Tal1/Lmo2 transgenic mice. Re-clustering of DN3 cells revealed the presence of dormant and proliferative DN3 cells in PL and Leuk thymus. Dormant DN3 signatures resembled quiescent, Notch1-active DN3a (pre-β-selection) thymocytes and proliferative DN3 cells were transcriptionally similar to DN3b thymocytes. Dormant mouse DN3 cells were enriched for signatures of quiescent, therapy resistant ALL patient cells and cells obtained from ALL patients at minimal residual disease (Ebinger et al., Cancer Cell 2016), suggesting that dormant leukemic DN3 cells resemble human L-ICs that mediate drug resistance and relapse. To test this, we employed in vivo nucleosome labeling to profile dormant DN3 cells throughout leukemia development and functionally evaluate their response to chemotherapy. To assess cell divisional history, WT, Tal1, Lmo2, or Tal1/Lmo2 mice harboring the tet-inducible H2B-GFP reporter allele were subjected to in vivo doxycycline pulse (labeling), followed by a two-week chase. Analysis of pulse-chased thymocytes revealed low frequencies of label retaining (GFPHI) DN3 cells in WT mice. However, label retention was significantly increased in Lmo2 DN3 thymocytes (2.9-fold), consistent with other Lmo2 models (Tremblay et al., Nat. Commun. 2018). Tal1 expression also promoted DN3 label retention (6.0-fold) and Tal1/Lmo2 co-expression resulted in further increases in DN3 GFPHI cells (25-fold), indicating that Tal1 and Lmo2 cooperate to promote DN3 dormancy. DN3 GFPHI cells were present at similar frequency in mice with overt T-ALL and in line with scRNA-seq data, GFPHI cells were observed nearly exclusively as a subset of the leukemic DN3a population. Treatment with Vincristine, DeXamethasone, and L-asparaginase (VXL) led to a further increase in GFPHI DN3 frequency in Lmo2 and Tal1 preleukemic mice (3.1- and 9.7-fold, respectively), suggesting that cell cycle restricted L-ICs may evade conventional chemotherapy and mediate relapse. We then tested whether dormancy is a feature of the L-IC in T-ALL patients. Analysis of relapsed pediatric T-ALL cells revealed that the CD7+CD1a- L-IC population (Chiu et al., Blood 2010) is cell cycle restricted compared to CD7+CD1a+ leukemic cells and that VXL treatment enriched for CD7+CD1a- cells in xenografted mice, suggesting that cell cycle restriction may be an important feature of both mouse and human T-ALL L-ICs. Analysis of transcriptomes to reveal mediators of dormancy that may be exploited to chemosensitize L-ICs will be discussed.

Humanized Nanobody Anti-CD72 CAR-T Cells Efficiently Eliminate B-Cell Malignancies Via Improved Affinity for CD72 but Induce Persistent Antigen Downregulation In Vivo

Background: Approximately 50% of pediatric B-cell acute lymphoblastic leukemia (B-ALL) and non-Hodgkin lymphoma patients treated with CD19 chimeric antigen receptor (CAR)-T cells relapse. CD22 CAR-T cells are curative only rarely. New immunotherapeutic targets are urgently needed. Our laboratory recently identified CD72 as a target on B-cell malignancies and developed a fully synthetic nanobody against CD72 (NbD4), demonstrating that NbD4 CAR-T cells (nanoCARs) are potent in vitro and in vivo (Nix et al., Cancer Discovery (2021)). We previously described the development of a humanized version of this nanobody binder (H24) that had improved efficacy in preliminary studies (Nix et al., ASH 2021). As we move CD72 nanoCARs toward clinical translation, in this follow-up study we aimed to compare H24 vs. NbD4 by 1) tumor clearance kinetics; 2) T-cell immunophenotypes; 3) in vivo mechanism of relapse; and 4) binding affinity to CD72. Methods: CD4/CD8 CAR-T cells were generated by lentiviral transduction. Single time point (24 hr) in vitro cytotoxicity was assessed across a range of effector:tumor (E:T) ratios. CAR-T elimination kinetics was determined by IncuCyte live cell imaging. CD45RA and CD62L flow cytometry was used to assess T-cell immunophenotypes. In vivo studies were performed by implanting 5e5 luciferase-labeled Jeko cells into NSG mice on Day -4, and administration of 3e6 CAR-T cells on Day 0. At sacrifice, flow cytometry was performed to evaluate CD72 expression on relapsed tumors. Relapsed Jeko was re-implanted into naïve NSG mice for 31 days. Binding affinity of recombinant NbD4 or H24 to biotinylated CD72 extracellular domain was determined by bio-layer interferometry. Results: In single time point assays, H24 nanoCARs eliminated SEM B-ALL cells with similar efficacy to CD19 CAR-Ts (not significant) and were more potent than NbD4 nanoCARs at a 1:10 E:T ratio (72% vs 44% cytotoxicity; p = 3e-4). Against a Jeko mantle cell lymphoma model, at a 1:1 E:T ratio, H24 nanoCARs were more potent than both CD19 CAR-Ts (96% vs 87%; p < 1e-4) and NbD4 nanoCARs (96% vs 70%; p < 1e-4). In live cell imaging assays, H24 nanoCARs eliminated SEM faster than CD19 CAR-Ts (Fig. 1a). After SEM co-culture, H24 nanoCARs secreted more IL-2 and IFNγ compared to NbD4 (IL-2 p < 1e-4; IFNγ p = 4e-4) and CD19 CAR-Ts (IL-2 p = 4e-4; IFNγ p = 3e-4). Versus SEM, both pre- and post-tumor proportions of naïve T cells (CD45RA+/CD62L+) were similar for H24, NbD4, and CD19 CAR-Ts. Bulk RNA-seq after exposure to SEM revealed that H24 nanoCARs upregulate genes relevant to T cell activation and effector T cell function, compared to CD19 CAR-Ts. In in vivo Jeko studies, we treated mice with NbD4 or H24 nanoCARs and extracted tumors after relapse. We found that both nanoCARs led to CD72 loss on tumors (Fig. 1b). Surprisingly, however, upon re-implantation into a new cohort of NSG mice, H24 relapse Jeko continued to maintain low CD72 expression, while re-implanted NbD4 relapse Jeko had restoration of CD72 in the absence of anti-CD72 immune pressure (H24 vs NbD4 CD72 expression p = 4e-2). Given more potent anti-tumor efficacy and more persistent CD72 suppression in murine models, we hypothesized that H24 has a higher binding affinity for CD72. Indeed, H24 demonstrated a dissociation constant (KD) versus CD72 of 33.0 ± 0.04 nM, while NbD4 was 50.4 ± 0.05 nM. These results support the notion that relatively low-affinity binders can still generate potent CAR-Ts, when compared to published values of KD <1 nM for the anti-CD19 scFv FMC63. Conclusions: We have developed a new, humanized anti-CD72 cellular therapy known as H24 nanoCARs. Humanizing the framework regions, while keeping the complementary determining regions (CDRs) identical to NbD4, was intended to reduce immunogenicity. We were surprised that eight amino acid substitutions in the framework regions, with no changes to CDRs, improved antitumor efficacy and binding affinity for CD72. These improved characteristics motivate our planned clinical translation of H24-based nanoCARs. We also found that CD72 downregulation occurs after CD72 nanoCAR treatment in mice, and that increased potency of H24 correlates with longer-term CD72 suppression on tumors. These findings will inform expected outcomes in future clinical trials and motivate further studies to prevent CD72 downregulation after nanoCAR treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Spectrum of Genetic Mutations in Korean Pediatric Acute Lymphoblastic Leukemia.

The wide application of next-generation sequencing (NGS) technologies has led to the discovery of multiple genetic alterations in pediatric acute lymphoblastic leukemia (ALL). In this work, we aimed to investigate the mutational spectrum in pediatric ALL. We employed a St. Mary’s customized NGS panel comprising 67 leukemia-related genes. Samples were collected from 139 pediatric ALL patients. Eighty-five patients (61.2%) harbored at least one mutation. In B-cell ALL, the RAS pathway is the most involved pathway, and the three most frequently mutated genes were NRAS (22.4%), KRAS (19.6%), and PTPN11 (8.4%). NRAS and PTPN11 were significantly associated with a high hyperdiploidy karyotype (p = 0.018 and p < 0.001, respectively). In T-cell ALL, the three most frequently mutated genes were NOTCH1 (37.5%), FBXW7 (16.6%), and PTEN (6.2%). Several pairs of co-occurring mutations were found: NRAS with SETD, NRAS with PTPN11 in B-cell ALL (p = 0.024 and p = 0.020, respectively), and NOTCH1 with FBXW7 in T-cell ALL (p < 0.001). The most frequent newly emerged mutation in relapsed ALL was NT5C2. We procured comprehensive genetic information regarding Korean pediatric ALL using NGS technology. Our findings strengthen the current knowledge of recurrent somatic mutations in pediatric ALL.

Predictive value of neutrophil-to-lymphocyte, lymphocyte-to-monocyte, and platelet-to-lymphocyte ratios in adult and pediatric acute lymphoblastic leukemia patients

Background Methods used for prognostication of acute lymphoblastic leukemia (ALL) are expensive; discovering low-cost prognostic factors is challenging. Objectives This study aimed to explore the prognostic role of baseline neutrophil-to-lymphocyte (NLR), lymphocyte-to-monocyte (LMR), and platelet-to-lymphocyte (PLR) ratios in predicting the response to end of induction chemotherapy in ALL patients. Patients and methods We included 44 adult patients and 47 pediatric patients who were newly diagnosed with ALL. All participants were subjected to a full history taking and a thorough medical examination. Laboratory investigations included complete blood count (CBC) with differential count analysis, with calculation of NLR, LMR, and PLR; bone marrow examination; conventional cytogenetic analysis; and immunophenotyping. Patients were followed until the end of the induction phase, and their response to treatment was assessed. Results Among the adult patients, 63.6% showed complete remission at the end of induction; their baseline CBC showed significantly lower NLR (P=0.001) and higher LMR (P=0.013). On the other hand, 66% of the pediatric patients showed good response to induction chemotherapy; their baseline CBC showed significantly lower NLR (P&lt;0.001), greater LMR (P=0.0134), and lower PLR (P=0.017). NLR more than or equal to 1, LMR less than or equal to 2.846, and PLR more than or equal to 39.1 were able to discriminate adult patients who will respond to induction chemotherapy, similarly NLR more than or equal to 1, LMR less than or equal to 3.286, and PLR more than or equal to 10 among pediatric patients. Conclusion Our research discovered that the rise in NLR and PLR, together with the decline of LMR at ALL diagnosis, could predict future resistance to the routinely used induction protocols, and the need for intensification regimens.

Optimizing thiopurine therapy in children with acute lymphoblastic leukemia: A promising "MINT" sequencing strategy and therapeutic "DNA-TG" monitoring.

Thiopurines, including thioguanine (TG), 6-mercaptopurine (6-MP), and azathioprine (AZA), are extensively used in clinical practice in children with acute lymphoblastic leukemia (ALL) and inflammatory bowel diseases. However, the common adverse effects caused by myelosuppression and hepatotoxicity limit their application. Metabolizing enzymes such as thiopurine S-methyltransferase (TPMT), nudix hydrolase 15 (NUDT15), inosine triphosphate pyrophosphohydrolase (ITPA), and drug transporters like multidrug resistance-associated protein 4 (MRP4) have been reported to mediate the metabolism and transportation of thiopurine drugs. Hence, the single nucleotide polymorphisms (SNPs) in those genes could theoretically affect the pharmacokinetics and pharmacological effects of these drugs, and might also become one of the determinants of clinical efficacy and adverse effects. Moreover, long-term clinical practices have confirmed that thiopurine-related adverse reactions are associated with the systemic concentrations of their active metabolites. In this review, we mainly summarized the pharmacogenetic studies of thiopurine drugs. We also evaluated the therapeutic drug monitoring (TDM) research studies and focused on those active metabolites, hoping to continuously improve monitoring strategies for thiopurine therapy to maximize therapeutic efficacy and minimize the adverse effects or toxicity. We proposed that tailoring thiopurine dosing based on MRP4, ITPA, NUDT15, and TMPT genotypes, defined as “MINT” panel sequencing strategy, might contribute toward improving the efficacy and safety of thiopurines. Moreover, the DNA-incorporated thioguanine nucleotide (DNA-TG) metabolite level was more suitable for red cell 6-thioguanine nucleotide (6-TGNs) monitoring, which can better predict the efficacy and safety of thiopurines. Integrating the panel “MINT” sequencing strategy with therapeutic “DNA-TG” monitoring would offer a new insight into the precision thiopurine therapy for pediatric acute lymphoblastic leukemia patients.

Implications of mitochondrial DNA variants in pediatric B-cell acute lymphoblastic leukemia

BackgroundResearch on the role of variations in the mitochondrial genome in pathogenesis of acute lymphoblastic leukemia (ALL) has been unfolding at a rapid rate. Our laboratory has previously described higher number of copies of the mitochondrial genomes per cell in pediatric ALL patients as compared to the healthy controls. In the current study, we evaluated the pattern of mitochondrial genome variations in 20 de-novo pediatric B-ALL cases and seven controls. Quantitative real-time Polymerase Chain Reaction was used for estimation of mitochondrial genomes’ copy number in bone marrow samples of each ALL patient and peripheral blood samples of controls. The complete mitochondrial genomes of all samples were sequenced using the Illumina platform.ResultsSequencing data analysis using multiple mitochondrial genome databases revealed 325 variants in all 27 samples, out of which 221 variants were previously known while 104 were unassigned, new variants. The 325 variants consisted of 7 loss-of-function variants, 131 synonymous variants, 75 missense variants, and 112 non-coding variants. New, missense variants (n = 21) were identified in genes encoding the electron transport chain complexes with most of them encoding ND4, ND5 of complex I. Missense and loss-of-function variants were found to be deleterious by many predictor databases of pathogenicity. MuTect2 identified true somatic variants present only in tumors between patient-sibling pairs and showed overlap with missense and loss-of-function variants. Online MtDNA-server showed heteroplasmic and homoplasmic variants in mitochondrial genome.ConclusionsThe data suggest that some of these variations might have a deleterious impact on the expression of mitochondrial encoded genes with a possible functional relevance in leukemia.

Relationship between Chemotherapy Side Effects on Gastrointestinal System and Quality of Life of Pediatric Acute Lymphoblastic Leukemia Patients

Relationship between Chemotherapy Side Effects on Gastrointestinal System and Quality of Life of Pediatric Acute Lymphoblastic Leukemia Patients

Investigating causes and risk factors of pre-chemotherapy viremia in acute lymphoblastic leukemia pediatric patients

BackgroundLeukemia patients are immune-compromised even before starting chemotherapy because the malignant cells invade the bone marrow and destroy WBC precursors. Leukemic patients are more susceptible to infection by a wide range of microorganisms. Viral infections and reactivations are common and may result in severe complications. The aim of this study is to investigate different causes of viremia in ALL pediatric patients as well as the clinical and the laboratory characteristics associated with viral infections.MethodsQualitative real-time PCR was used to detect (polyoma BK, parvo B19 and herpes simplex virus) DNA in the blood of ALL patients and routine hospital records were used to provide the data of hepatitis B & C virus infection.ResultsPolyoma BK was the most common detected virus (51.2%) followed by herpes simplex (30.2%). Viremia by single virus was found in 16 (37.2%) cases, while viremia by multiple viruses was found in 15 (34.8%) cases. The most frequent co-detected viruses were herpes simplex and polyoma BK (11.6%) followed by herpes simplex, parvo B19 and polyoma BK (9.3%).ConclusionThere is a high frequency of viremia by single virus and viremia by multiple viruses at the time of diagnosis of acute lymphoblastic leukemia in pediatric patients admitted to South Egypt Cancer Institute (SECI) compared to studies in other regions. Polyoma BK is the most common detected virus and is mainly associated with lymphopenia. It was also significantly associated with herpes simplex viremia. HCV infection was associated with increased incidence of CNS leukemia.

Efficacy and Safety of Levofloxacin Prophylaxis During Induction Therapy for Childhood Acute Lymphoblastic Leukemia

Background: Infection related complications represent an important cause of morbidity and mortality in pediatric cancer patients, especially in those receiving chemotherapy. Although antibiotic prophylaxis is used in adult leukemic patients, but it is less practiced in pediatric cancer patients. The aim of this study was to evaluate the efficacy and safety of Levofloxacin prophylaxis to reduce infection which occur during induction phase of chemotherapy among pediatric Acute Lymphoblastic Leukemia (ALL) patients.&#x0D; Materials and methods: This single centered, pilot study in RCT design was conducted in Pediatric HematoOncology Department of Chittagong Medical College Hospital (CMCH). Sixty newly diagnosed ALL patients admitted for chemotherapy were randomly allocated to two groups 30 in each: one group received Levofloxacin prophylaxis (Intervention group) and other group did not receive prophylaxis (Control group). During the whole period of induction, clinical and laboratory features of infection, fever and neutropenia were observed and adverse effects were noted in both groups.&#x0D; Results: In induction phase of chemotherapy of ALL, significantly lower proportion of patients developed fever in the intervention group than in the control group [(18/30, 60.00%) versus (26/30, 86.67%)]. Incidence of infection was significantly less in intervention group compared to control group (60% versus 86.7%, p=0.039). Also, incidence of febrile neutropenia was significantly less in intervention group compared to control group (p=0.039). No major adverse effects were observed in any of the levofloxacin prophylaxis group except only two patients reported mild lower limb pain. &#x0D; Conclusion: It may be concluded from this pilot study that, Levofloxacin prophylaxis in children with ALL during the induction phase of chemotherapy is effective in reducing infection, febrile episodes and febrile neutropenia.&#x0D; JCMCTA 2022 ; 33 (1) : 34-39

P360: EFFICACY AND SAFETY OF DARATUMUMAB IN PEDIATRIC AND YOUNG ADULT PATIENTS WITH RELAPSED/REFRACTORY T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA OR LYMPHOBLASTIC LYMPHOMA: RESULTS FROM PHASE 2 DELPHINUS STUDY

Background: Approximately 15-20% of pediatric patients with T-cell acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LL) will be refractory to/relapse after frontline treatment; relapsed disease is associated with poor outcomes. In a phase 2 study, 2 of 7 (28.6%) patients with T-cell ALL in first relapse achieved a complete response (CR) using the vincristine, prednisone, PEG-asparaginase, and doxorubicin (VPLD) reinduction backbone. Daratumumab (DARA), a human IgGκ monoclonal antibody targeting CD38 approved for treating multiple myeloma, has shown preclinical efficacy in ALL models. Aims: Here we report the initial results of DARA plus VPLD in pediatric and young adult patients with relapsed/refractory T-cell ALL or LL enrolled in the phase 2, open-label DELPHINUS study. Methods: Eligible patients were aged 1-30 y, had T-cell ALL or LL in first relapse or refractory to 1 prior induction/consolidation regimen, and had a performance status ≥70. DARA (16 mg/kg IV QW) was given with VPLD in Cycle 1 and with methotrexate, cyclophosphamide, cytarabine, and 6-mercaptopurine in Cycle 2. Patients received age/risk-adjusted intrathecal therapy. Response was measured at the end of each cycle by local bone marrow morphology. The primary endpoint for ALL patients was CR rate in pediatric patients at the end of Cycle 1. Patients achieving CR after Cycles 1 or 2 could proceed to allogeneic HSCT off study. Overall response rate (ORR) was defined as CR or CR with incomplete hematological recovery (CRi) at any time before start of subsequent therapy or HSCT. Minimal residual disease (MRD) negativity (<0.01%) at any time before disease progression, start of subsequent therapy, or HSCT was centrally reviewed by flow cytometry. Results: Twenty-four pediatric (age 1-17 y) and 5 young adult (age 18-30 y) ALL patients and 10 LL patients (age 1-30 y) received ≥1 DARA dose. Median (range) age was 10.0 (2-25) y (ALL) and 14.5 (5-22) y (LL); median (range) time from initial diagnosis to first study treatment was 2.0 (0.1-6.1) y (ALL) and 0.8 (0.5-6.0) y (LL). Pediatric ALL patients received a median (range) of 2 (1-3) treatment cycles; young adult ALL patients and LL patients each received 2 (1-2). Among pediatric ALL patients, 10 (41.7%) patients (90% CI, 24.6-60.3) achieved CR at the end of Cycle 1. ORR was 83.3% (CR, 13 [54.2%] patients; CRi, 7 [29.2%] patients) in pediatric and 60.0% (all CR) in young adult ALL patients and 40.0% (all CR) in LL patients. Ten (41.7%) pediatric ALL patients achieved MRD negativity. All pediatric ALL patients had a grade 3/4 TEAE. No pediatric ALL pt discontinued DARA primarily due to AEs and 1 (4.2%) died due to TEAEs (brain edema and hepatic failure) attributed to study treatment but unrelated to DARA. Summary/Conclusion: The addition of DARA to VPLD in pediatric and young adult patients with relapsed/refractory T-cell ALL or LL showed initial activity, generating improved response rates compared to those achieved with backbone therapy alone, with a manageable safety profile.

Enrichment of cancer-predisposing germline variants in adult and pediatric patients with acute lymphoblastic leukemia

Despite recent progress in acute lymphoblastic leukemia (ALL) therapies, a significant subset of adult and pediatric ALL patients has a dismal prognosis. Better understanding of leukemogenesis and recognition of germline genetic changes may provide new tools for treating patients. Given that hematopoietic stem cell transplantation, often from a family member, is a major form of treatment in ALL, acknowledging the possibility of hereditary predisposition is of special importance. Reports of comprehensive germline analyses performed in adult ALL patients are scarce. Aiming at fulfilling this gap of knowledge, we investigated variants in 93 genes predisposing to hematologic malignancies and 70 other cancer-predisposing genes from exome data obtained from 61 adult and 87 pediatric ALL patients. Our results show that pathogenic (P) or likely pathogenic (LP) germline variants in genes associated with predisposition to ALL or other cancers are prevalent in ALL patients: 8% of adults and 11% of children. Comparison of P/LP germline variants in patients to population-matched controls (gnomAD Finns) revealed a 2.6-fold enrichment in ALL cases (CI 95% 1.5–4.2, p = 0.00071). Acknowledging inherited factors is crucial, especially when considering hematopoietic stem cell transplantation and planning post-therapy follow-up. Harmful germline variants may also predispose patients to excessive toxicity potentially compromising the outcome. We propose integrating germline genetics into precise ALL patient care and providing families genetic counseling.

Total body irradiation versus chemotherapy conditioning in pediatric acute lymphoblastic leukemia patients undergoing hematopoietic stem cell transplant: A systematic review and meta-analysis.

e22002 Background: Hematopoietic stem cell transplant (HSCT) is indicated in pediatric acute lymphoblastic leukemia (ALL) patients who have relapsed or are at a very high risk of relapse during first complete remission. Two types of myeloablative conditioning have been employed before HSCT: total body irradiation (TBI) based regimens and chemotherapy alone (CHT). The use of TBI is favorable in adults with ALL, and it has also demonstrated excellent efficacy in the pediatric population. However, the lifelong adverse events of TBI are a substantial concern in younger patients. This study compares the efficacy and safety of TBI and CHT conditioning in pediatric, adolescent and young adult ALL patients (0-24 years old). Methods: A systematic literature search was conducted on PubMed, Embase, Cochrane, and Clinicaltrials.gov from inception to November 25, 2021, using MeSH terms and keywords for ‘acute lymphoblastic leukemia’, ‘whole body irradiation’, ‘child’, ‘adolescent’ and ‘young adult’. Efficacy outcomes considered were overall survival (OS), event-free survival (EFS), and relapse. Safety outcomes were acute (a) and chronic (c) graft-versus-host disease (GVHD), and non-relapse mortality/transplant-related mortality (NRM/TRM). Estimates of risk ratios (RR) for dichotomous variables and hazard ratios (HR) for OS and EFS were pooled using a random-effects model in Revman v5.4. Results: The initial search revealed 1544 articles. After excluding reviews, duplicates, and non-relevant articles, we included data from 16 studies (2 randomized controlled trials, 1 non-randomized prospective study and 13 retrospective studies). TBI based and CHT conditioning were evaluated in 4656 and 1500 patients, respectively. The most common TBI and CHT regimens were TBI + cyclophosphamide (Cy) + etoposide (3 studies) and busulfan + Cy (6 studies), respectively. The TBI dose utilised ranged from 8-12 Gy, with 12 Gy being the most common (8 studies). The median age ranged from 4 months to 13.3 years; and the median follow up was 1.2-9.7 years. In comparison with TBI conditioning, CHT alone was associated with significantly worse OS (HR 1.61, 95% CI 1.06-2.44; p = 0.03; I2 = 79%), EFS (HR 2.01, 95% CI 1.26-3.20; p = 0.003; I2 = 60%) and risk of relapse (RR 1.44, 95% CI 1.23-1.68; p = 0.00001; I2 = 37%). There was reduced risk of aGVHD with CHT (RR 0.79, 95% CI 0.66-0.93; p = 0.006; I2 = 51%), but the difference was not significant when only grade 3-4 aGVHD was considered (RR 1.01, 95% CI 0.80-1.27; p = 0.95; I2 = 0%). The two regimens were comparable in terms of risk of cGVHD (RR 0.87, 95%CI 0.64-1.18; p = 0.38; I2 = 43%) and NRM/TRM (RR 1.24, 95% CI 0.99-1.55; p = 0.06; I2 = 66.8%). Conclusions: In pediatric and young adult ALL patients undergoing HSCT, TBI conditioning has better efficacy and similar safety compared to CHT alone.

Measurable residual disease analysis in paediatric acute lymphoblastic leukaemia patients with ABL-class fusions

BackgroundABL-class fusions including NUP214-ABL1 and EBF1-PDGFRB occur in high risk acute lymphoblastic leukaemia (ALL) with gene expression patterns similar to BCR-ABL-positive ALL. Our aim was to evaluate new DNA-based measurable residual disease (MRD) tests detecting these fusions and IKZF1-deletions in comparison with conventional immunoglobulin/T-cell receptor (Ig/TCR) markers.MethodsPrecise genomic breakpoints were defined from targeted or whole genome next generation sequencing for ABL-fusions and BCR-ABL1. Quantitative PCR assays were designed and used to re-measure MRD in remission bone marrow samples previously tested using Ig/TCR markers. All MRD testing complied with EuroMRD guidelines.ResultsABL-class patients had 46% 5year event-free survival and 79% 5year overall survival. All had sensitive fusion tests giving high concordance between Ig/TCR and ABL-class fusion results (21 patients, n = 257 samples, r2 = 0.9786, P < 0.0001) and Ig/TCR and IKZF1-deletion results (9 patients, n = 143 samples, r2 = 0.9661, P < 0.0001). In contrast, in BCR-ABL1 patients, Ig/TCR and BCR-ABL1 tests were discordant in 32% (40 patients, n = 346 samples, r2 = 0.4703, P < 0.0001) and IKZF1-deletion results were closer to Ig/TCR (25 patients, n = 176, r2 = 0.8631, P < 0.0001).ConclusionsMRD monitoring based on patient-specific assays detecting gene fusions or recurrent assays for IKZF1-deletions is feasible and provides good alternatives to Ig/TCR tests to monitor MRD in ABL-class ALL.