Newly-diagnosed Acute Lymphoblastic Leukemia Research Articles

Trends of Obesity over Time and Its Prognostic Significance in Children and Adolescents with Newly-Diagnosed Acute Lymphoblastic Leukemia in Canada: A Population-Based Study

Introduction Childhood obesity can result in a variety of adverse health outcomes and it has been associated with inferior survival in children and adolescents with acute lymphoblastic leukemia (ALL). With the rising incidence of childhood obesity in Canada, we seek to assess the prevalence of obesity and its prognostic significance in children and adolescents with newly-diagnosed ALL in Canada. Methods We conducted a retrospective cohort study using the Cancer in Young People in Canada (CYP-C) database, a national population-based pediatric surveillance program collecting in-depth clinical data from all cancer patients <15 years old before 2015 and <19 years old after 2015 in Canada. This study included patients aged 2 to 18 years old with newly-diagnosed ALL and treated at one of the 17 pediatric oncology centers across Canada from January 1, 2001 to December 31, 2020. Obese patients were defined as patients with a body mass index (BMI) at or greater than the 95 th age and sex-adjusted percentile as per the Centers for Disease Control & Prevention (CDC). Patients <2 years old were excluded given no BMI data could be derived from CDC growth charts. The main outcomes were event-free survival (EFS) and overall survival (OS). EFS was defined as time from ALL diagnosis to first event (relapse or death). OS was defined as time from ALL diagnosis to death from any cause. Patients without an event were censored at date of last contact. Predictors of obesity were explored using logistic regression. Univariate and multivariable Cox proportional hazards models were used to compare EFS and OS between patients with and without obesity. Results A total of 3458 ALL patients were included. The median BMI was 16.4 kg/m 2 (interquartile range (IQR): 15.2-18.1 kg/m 2). Eleven percent of children were obese at the time of ALL diagnosis. The prevalence of obesity significantly increased over time compared to that at ALL diagnosis: 18.9% at 1 year (p<0.0001), 25.8% at 2 years (p<0.0001), 23.3% at 3 years (p<0.0001), 21.8 % at 4 years (p<0.0001) and 20% at 5 years (p<0.0001). The prevalence of obesity did not vary according to age at diagnosis, gender, neighborhood income quintile (NIQ), ALL immunophenotype and treatment era ( Table 1). Non-Caucasian race (odds ratio (OR): 1.35 [95% confidence interval (CI) 1.09 -1.67]; p=0.005), presenting white blood cell (WBC) ≥ 50 x 10 9/L (OR: 1.38 [1.07 - 1.77]; p=0.01) and central nervous disease (CNS) disease (OR: 1.44 [1.10 - 1.88]; p=0.009) were significantly associated with increased risk of obesity at diagnosis. The 5-year EFS of obese patients at ALL diagnosis were significantly inferior to those of non-obese patients (85.3% vs. 90.6%; p=0.0008) ( Figure 1). The 5-year OS was 90.4% for obese vs. 94.9% for non-obese patients at diagnosis (p=0.003). The causes of death in the entire cohort were 1) related to disease (73.5%); 2) related to treatment toxicities (15.6%); and 3) due to other causes (10.9%). In univariate analysis, obese patients at ALL diagnosis had a higher risk for worse EFS (Hazard Ratio (HR) 1.76, 95% CI 1.34 - 2.32, p<0.0001)and OS (HR 1.99, 95% CI 1.41- 2.81, p<0.0001) compared to non-obese patients. In addition, age ≥ 10 years old at diagnosis, CNS disease, presenting WBC count ≥ 50x10 9/L, non-favorable cytogenetics and treatment era before 2010 were also significantly associated with worse EFS and OS in univariate analyses. In multivariable models controlling for patients' age at diagnosis, gender, presenting WBC, CNS status, NIQ, ethnicity, treatment era and ALL immunophenotype, obesity at ALL diagnosis retained independent significance with a HR of 1.67, 95% CI 1.26-2.21, p=0.0004 for EFS and a HR of 1.83, 95% CI 1.29-2.61, p=0.0008 for OS. Conclusion In this population-based study of children and adolescents with ALL in Canada, obesity was found in 11% of patients at ALL diagnosis, peaked at 2 years and remained significantly increased at 5 years from initial diagnosis. Patients with higher risk features such as WBC ≥ 50 x 10 9/L and CNS disease were more likely to be obese at diagnosis. Nevertheless, obesity at ALL diagnosis remained independently associated with inferior EFS and OS even after adjustment for these high-risk features. More mechanistic studies are needed to better understand the relationship between obesity and outcome to inform future trial designs.

Delaying Pegaspargase Administration during Induction in Adults with Acute Lymphoblastic Leukemia

Background Pegaspargase (PEG-asparaginase) is a key drug in pediatric-inspired regimens that is utilized frequently in young adults with acute lymphoblastic leukemia (ALL). Pegaspargase has unique toxicities including hypersensitivity, hepatotoxicity, pancreatitis, thrombosis and hypertriglyceridemia. Hepatotoxicity occurs frequently during the induction cycle in newly diagnosed (ND) adults with ALL, and although it is usually reversible, its prolonged persistence may cause interruptions in the treatment. We analyzed two approaches for pegaspargase administration in ND adults with ALL; early administration on day 4 and delayed administration on day 15 that were utilized in our treatment protocols.We hypothesized that later administration of pegaspargase is associated with lower risk of toxicity while maintaining similar response rate. Methods This is a retrospective analysis of adult patients (age:19-71) with ND ALL that received pegaspargase during their first induction chemotherapy cycle. Patients were stratified into two groups, the early (day 4) and delayed (day 15) pegaspargase group. The primary objective was to assess differences in toxicities, including hepatotoxicity, hypertriglyceridemia, thrombosis, and pancreatitis. Adverse events were graded using NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. Key secondary outcomes included differences in minimal residual disease (MRD), complete remission (CR) rates, and induction related mortality. Statistical analysis was done with descriptive statistics, Mann Whitney U test, Pearson chi-squared test, and multivariate analysis (p-value < 0.05). Results Of 117 patients analyzed, there were 72 (62%) patients who received early pegaspargase (EP) while 45 (38%) patients received delayed pegaspargase (DP). The median age of patients was younger in the EP cohort (31.5 vs. 38, p=0.03) and there was no significant difference in gender (p=0.36), BMI (p=0.42), or median pegaspargase dose (p=0.12). CALGB10403 was the most common chemotherapy regimen in the EP cohort and in 20% of DP cohort, while modified BFM was the most common chemotherapy regimen in DP cohort (80%). Grade 3/4 transaminitis occurred more frequently in EP compared to DP cohort (15% vs 2%; p=0.02), however, other grade 3/4 toxicities were not statistically different: hyperbilirubinemia (21% vs 11%; p=0.17), hypertriglyceridemia (3% vs 9%; p=0.14), and pancreatitis (4% vs 2%; p=0.57), respectively. The CR rate (88% vs 80%; p= 0.18) and end of induction MRD negative rate (60% vs 61%; p=0.86) were similar for EP and DP cohorts, respectively. There was no significant difference in the rates of documented infections during induction (10% vs 9%; p=0.88) and 90-day mortality (2% vs 0%; p=0.42) between EP and DP cohorts, respectively. Post-pegaspargase dose modifications to induction chemotherapy were only observed in the EP cohort (18% vs 0%; p=0.002), with modifications including reduction (5%), delays (6%), and discontinuation (5%) of other chemotherapy agents. In multivariable logistic regression analysis, only older age (OR 1.05; p=0.025) and EP administration (OR 4.58; p=0.007) predicted increased risk of grade 3/4 hepatotoxicity (transaminitis and/or hyperbilirubinemia). Conclusions Delaying pegaspargase administration from day 4 to day 15 during induction cycle in adults with ALL was associated with lower rate of high-grade hepatotoxicity and subsequent treatment dose modifications. Delaying pegaspargase had no negative impact on CR or MRD-negativity rates at the end of induction.

Differences in induction phase outcomes of acute lymphoblastic leukemia between well-nourished and malnourished pediatric patients

Background Leukemia is the most common malignancy in childhood. Malnutrition is the main nutritional disorder occurring in children with cancer. Nutritional supportive care is a medical modality that has been associated with improved tolerance to chemotherapy, survival, and quality of life, as well as decreased risk of infection in children undergoing cancer therapy.
 Objective To examine differences in induction phase outcomes in well-nourished and malnourished pediatric acute lymphoblastic leukemia (ALL) patients.
 Methods This prospective study was conducted in newly-diagnosed ALL patients aged 1 to 18 years who underwent induction phase chemotherapy at Haji Adam Malik General Hospital, Medan, North Sumatera. Mid-upper arm circumference (MUAC) was used to determine nutritional status. Patient characteristics such as complete blood count (CBC) at the time of diagnosis, occurrence of febrile neutropenia, duration of induction, length of hospital stay, and remission status were collected. We analyzed differences in CBC, frequency of febrile neutropenia, as well as duration of induction and hospital stay between well-nourished and malnourished patients. We also assessed the difference in remission attainment between the two groups.
 Results There were 21 subjects in each group. Compared to well-nourished subjects, malnourished ones had a higher median incidence of febrile neutropenia [1 (range 0 to 30) vs. 3 (range 0 to 4); P=0.04], longer mean induction duration [64.2 (SD 11.5) vs. 71.2 (SD 10.6) days; P=0.046], and longer median hospital stay [30 (range 19 to 56) vs. 36 (range 22 to 49) days; P=0.001] compared to well-nourished subjects. There was no significant difference in CBC parameters and remission status after induction between the two groups.
 Conclusion Malnourished pediatric ALL patients had a significantly higher incidence of febrile neutropenia, duration of induction phase, and duration of hospital stay compared to well-nourished ALL patients.

Phase II study of dose-adjusted EPOCH as initial therapy for adults with high-risk acute lymphoblastic leukemia

Treatments for adults with newly-diagnosed acute lymphoblastic leukemia (ALL) may be prohibitively toxic and/or resource-intense. To address this, we performed a phase II study of dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (DA-EPOCH). Imatinib or dasatinib was added for Ph + disease; rituximab was added when CD20+. Fifty-three patients were evaluable: 28 with Ph + disease, and 25 with Ph−. All patients had ≥1 high-risk clinical feature. Measurable residual disease-negativity by multiparameter flow cytometry within 4 cycles was achieved in 71% in patients with Ph + ALL and 64% in Ph − ALL. Median overall survival (OS) was 49 months, with a 2-year OS of 71%. Median relapse-free survival (RFS) in the 47 patients that attained morphologic remission was 24 months, with a 2-year RFS of 57%. Early mortality was 2%. In summary, DA-EPOCH yields deep and durable remissions in adults with ALL comparable to some resource-intense strategies but with a low rate of treatment-related death.

Experience of Paediatric Acute Lymphoblastic Leukemia Service in A Newly Established Haemato-Oncology Center in Bangladesh: Opportunities and Challenges

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy and its modern management is complex. We are reporting early experience of establishing a paediatric ALL service in a multidisciplinary paediatric hospital in Bangladesh.
 Methods: This is a retrospective review of children below 18 years of age with confirmed diagnosis of ALL from July 2020 to June 2021 in Dr. M R Khan Shishu Hospital and ICH, Dhaka, Bangladesh, who received treatment adapted from the standard arm of ICICLE (Indian Childhood Collaborative Leukemia Group) protocol. Data were collected which included demographic, clinical, laboratory features of all patients at the time of presentation and also morbidities and outcome during all phases of chemotherapy. Analysis was done using descriptive statistics.
 Results: Of 51 patients, 16 were newly diagnosed patients, 32 received continuation care and 3 had relapsed disease. Treatment was initiated in 12 (75%) of 16 patients with newly-diagnosed ALL. Median age was three years, 50% were girls, one had TALL and 5 (42%) had high presentation leucocyte count (³50,000/mm3). Complete cytogenetic testing was available for one patient alone, no patient had Ph+ ALL. Eleven (92%) showed good prednisolone response. All nine patients who completed the induction phase achieved morphological remission, with high minimal residual disease (³0·01%) in two (22%). At last follow-up (30-06-2021), two patients were midway through induction, two died from sepsis (one each in Induction and Consolidation, both high risk ALL) and eight (67%) are alive in remission, on treatment 2-12 months from diagnosis. Continuation care included intrathecal treatments (n=119) and vincristine-corticosteroid pulses (n=53); 94% patient remained in complete remission, while two (6%) relapsed during the course of treatment.
 Conclusion: Risk-stratified ALL treatment is feasible in a newly established resource limited setting but limited by availability of high-quality diagnostics, specifically cytogenetics. Our study revealed that, during intensive phase approximately two-third children and during maintenance phase majority of children remained in complete remission.
 DS (Child) H J 2021; 37(2): 116-122

Prognostic Significance of Chromosomal Genomic Array Testing in Adults with Newly Diagnosed Acute Lymphoblastic Leukemia

Introduction: As part of the diagnostic workup in newly diagnosed acute lymphoblastic leukemia (ND-ALL) in adults, chromosomal genomic array testing (CGAT) is frequently added to karyotype and fluorescence in situ hybridization (FISH) to enhance mutational profiling by allowing for more precise analysis of genomic copy number alterations (CNAs) and copy-neutral loss-of-heterozygosity that may impact genes of clinical significance. Despite its increasing use, the prognostic role of CGAT in ND-ALL in adults, and how to apply its findings to clinical decision making, is not well-established. Stanulla and others (JCO 2018) defined the IKZF1plus signature as IKZF1 deletions co-occurring with deletions in CDKN2A/B, PAX5, or PAR1 in the absence of an ERG deletion: this was based on CNAs and carries adverse prognostic significance in pediatric ALL. We hypothesized that by correlating CGAT results at the time of diagnosis with key demographic and outcomes data, we can enhance our ability to risk-stratify patients (pts) at the time of diagnosis. Methods: Eligible pts included those ≥18 years old diagnosed with ALL at our center between January 2012 and March 2022. We collected relevant demographic, laboratory, treatment, and outcome data. For CGAT, genomic DNA (gDNA) was processed on the CytoScan™ HD Array (ThermoFisher, MA), targeting a genome-wide region with 2.4 million markers for copy number and approximately 750,000 genotype-able small nucleotide polymorphisms (SNPs). The data were analyzed using Nexus Copy Number software with manual curation. Karyotype and FISH were performed in the conventional manner and classified per NCCN. The primary objectives were to characterize recurrent CGAT abnormalities and examine their correlation with survival. Results: We identified 196 pts with ND-ALL who had CGAT testing performed at diagnosis and received treatment at our center: 54 pts with available data comprised our initial discovery set (Table). CGAT among these pts disclosed large segmental CNAs frequently associated with ALL, such as gains of chromosome X, 1/1q, and 8q; and deletions of 7p, 9p, and 12p. Peak regions with CNAs were detected, which are regions with higher frequencies of aberrations compared with chromosomal segments on either side. We further investigated peak regions that showed aberrations in at least 15% of pts. After removing those that are entirely copy number variation (CNV), the following genomic loci were identified, in order of decreasing frequency: IKZF1 (55%; 17% homozygous loss), CDKN2A (50%), PAX5 (26%), ZCCHC7 (24%), LINC01619 (24%), RB1 (19%), ETV6 (17%), EBF1 (15%), ATP10A (15%), and SLX4IP (15%). The IKZF1plus signature was identified in 30% of pts. In pts without high-risk cytogenetics (n=17), 88% harbored aberrations in ≥1 of these genes: 29% had IKZF1 deletion and 18% had the IKZF1plus signature. Other commonly observed associations include IKZF1 deletion seen in 85% of Philadelphia-chromosome (Ph)+ pts (n=20) vs. 38% of Ph- (n=34) and CDKN2A deletion seen in 77% of pts with WBC >30K/µL (n=18) vs. 39% without (n=36). With a median follow-up of 2.1 years, there was no significant difference in median OS of the IKFZ1plus cohort (n=17; 4.1 yrs) vs. all IKZF1-deleted (including mono- and biallelic deletions) cohort (n=30; 4.1 yrs), vs. IKZF1 wild type (WT) (n=24, not reached) (p = 0.7). We next created univariate logistic regression models for mortality: statistically significant genomic loci included PAX5 deletion (OR 5.26, 95% CI 1.47-20.0), ZCCHC7 deletion (OR 4.34, 95% CI 1.17-16.67), and ETV6 deletion (OR 5.0, 95% CI 1.07-22.7). In a multivariable logistic regression model for mortality, the strongest associations were seen with PAX5 deletion (OR 4.0, 95% CI 1.0-16.39) and ETV6 deletion (OR 5.0, 95% CI 1.1-27.78): the median OS in the PAX5 deletion (n=14; 2.2 yrs) and the ETV6 deletion (n=9; 2.2 yrs) cohorts was inferior to the PAX5/ETV6 WT cohort (n=35; not reached) (p=0.035; Figure). Discussion: In this initial discovery-phase analysis of CGAT in ND-ALL in adults, we identified CNAs involving well-described genes (e.g. IKZF1), and lesser-described genes (e.g. LINC016919). Abnormal CGAT was seen in nearly all pts with otherwise normal or good cytogenetic risk. IKZF1plus was not associated with worse survival in this adult cohort, though deletions of PAX5 and ETV6 were. Analysis of additional pts to validate the significance of our findings will be presented. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Sperm Banking in Post-Pubertal Males with Newly Diagnosed High-Risk ALL

Purpose: 10-15% of adolescent and young adults (AYA) with high-risk pre-B acute lymphoblastic leukemia (ALL) may need treatment for relapsed/refractory leukemia, leading to impaired fertility. While hospitalized, sperm banking may be difficult for a newly-diagnosed AYA with ALL. This study examined practices and specific barriers to offering sperm banking at initial ALL diagnosis prior to starting chemotherapy. Methods: This cross-sectional survey study was distributed by email to all Children's Oncology Group (COG) oncologists (n=2345) in the United States, Canada, Australia and New Zealand from January-April 2022. Data were collected regarding size of program and institutional practices for sperm banking. Program size was defined by the total numbers of new ALL diagnoses per year: small (<10 per year), medium (10-30) or large (>30 per year). Data were summarized with counts and percentages and compared between groups using chi-square tests or Fisher's exact tests. Results: 572 surveys were completed, representing a 25% participation rate. 78% of responders offer sperm banking to physically able males with newly-diagnosed ALL prior to starting chemotherapy. Commonly reported barriers were: insufficient time before chemotherapy (70%), patients being too sick (44%), and lack of perceived need since most will be fertile after initial treatment (41%). 57% of the total responders work at an institution with a dedicated oncofertility team. Of the 445 responders who offer sperm banking, 190 (43%) work at programs without a dedicated oncofertility team. Most (86%) responders who offer sperm banking work at medium/large-sized institutions. Conclusion: A majority of COG oncologists who responded to our survey offer sperm banking prior to starting chemotherapy. The most frequently-cited barriers included insufficient time before treatment and patients being too sick. It is encouraging that 43% of individuals who offer sperm banking work at institutions without a dedicated oncofertility team. Medium/large institutions may have more resources to facilitate sperm banking in this setting. A possible study limitation may be respondent-bias towards individuals passionate about fertility preservation who have already adopted the practice of sperm banking at diagnosis and are not representative of all pediatric oncologists. Future directions include obtaining additional insight into COG oncologists who did not respond in order to improve education and best practices around offering sperm banking to AYA with newly-diagnosed high-risk ALL regardless of institution size or dedicated fertility preservation team. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparison of Overall Survival Among Adolescents and Young Adults (AYA) with Newly-Diagnosed Acute Lymphoblastic Leukemia (ALL) Treated with Pediatric-Inspired or Non-Pediatric Inspired Regimens: A Real-World Analysis Using Claims Data

Comparison of Overall Survival Among Adolescents and Young Adults (AYA) with Newly-Diagnosed Acute Lymphoblastic Leukemia (ALL) Treated with Pediatric-Inspired or Non-Pediatric Inspired Regimens: A Real-World Analysis Using Claims Data

Thrombin Generation Is Differentially Affected By Anticoagulants in Patients with Acute Lymphoblastic Leukemia - an Ex-Vivo Study

Background: Venous thromboembolism (VTE) is a frequent complication in patients with acute lymphoblastic leukemia (ALL). Low molecular weight heparin (LMWH) reduces the risk of VTE in ALL, but is not well tolerated in children. VTE rates remain unacceptably high in adults despite LMWH prophylaxis. Thus, novel strategies for VTE prevention are warranted. In the primary analysis of the PREVAPIX-ALL trial there was no statistically significant reduction in VTE among children treated with the oral factor Xa inhibitor, apixaban, compared to no anticoagulation (AC). Comparative data on activity of different anticoagulants are needed to guide planning of VTE prophylaxis trials. Aims: To characterize and compare the ex-vivo effect of LMWH (enoxaparin), oral direct factor Xa inhibitors (apixaban, rivaroxaban) and oral direct thrombin inhibitor (dabigatran etexilate) in ALL patients' samples using thrombin generation (TG). Methods: A prospective observational cohort study of 46 children with newly-diagnosed ALL, treated with the L-asparaginase (ASP)-based BFM protocol, at a tertiary medical center. Patients on therapeutic-dose AC at baseline were excluded. Blood samples were drawn before ASP treatment (ALL-Day0) and 7 days after (ALL-Day7). Normal pooled plasma (NP) was collected from healthy adult volunteers. Samples were spiked ex-vivo with enoxaparin [0.2 U/mL], apixaban [39 ng/mL], rivaroxaban [39 ng/mL] and dabigatran [150 ng/mL] or vehicle (i.e., control). AC concentrations chosen based on 50% peak reduction (for Xa inhibitors) or 200% LT prolongation (for dabigatran) in NP. Subsequently, TG was performed initiated using 5 pM tissue factor in the presence of 4 µM phospholipids using Calibrated Automated Thrombogram method in these samples. Results are expressed as peak height (PH) and lag time (LT) and relative reduction or prolongation (% difference in PH or LT, respectively) in plasma samples spiked with anticoagulants compared to control. Reduction in PH reflects enoxaparin, apixaban and rivaroxaban activity, while LT prolongation indicates dabigatran activity. This report focuses on measures reflecting the activity of each drug. Descriptive statistics were used to show the median [interquartile range (IQR)] for continuous variables and percentages for categorical data. T-Test / Mann-Whitney U test and paired T-test / Wilcoxon test used to compare between paired and non-paired samples, respectively. Results: Selected sample characteristics are as follows: median age, 7.75 years [IQR 3.73-11.93]; 19 (41%) females; median BMI, 16.8 [IQR 15.5-19.9]; B-cell precursor immunophenotype in 33 (71.7%) and T-cell in 13 (28.3%); prior VTE in 1 (2%). Median PH [IQR] in control samples (without AC) was 256.6 [252.6-265.3] nmol/l, 396.0 [347.2-464.7] nmol/l and 354.4 [307.1-407.4] nmol/l in NP, ALL-Day0 and ALL-Day7 respectively. Median LT [IQR] in control samples was 2.7 [2.5-2.83] nmol/l, 1.8 [1.7-2.3] nmol/l and 1.7 [1.6-2] nmol/l in NP, ALL-Day0 and ALL-Day7 respectively. Median % differences in PH and LT between samples spiked with different anticoagulants and controls are shown in Figure 1A and Figure 1B, respectively. Enoxaparin, apixaban and rivaroxaban reduced PH in NP, ALL-Day0 and ALL-Day7 samples (p<0.0005). There was less reduction in PH with enoxaparin, apixaban and rivaroxaban in ALL-Day0 samples than in NP (p=0.029, p<0.0005, p<0.0005 respectively), suggesting reduced anticoagulant activity in ALL patients compared to subjects without ALL. LT was prolonged with dabigatran in NP, ALL-Day0 and ALL-Day7 samples (p<0.05). Dabigatran resulted in a similar LT prolongation in ALL-Day0 and NP (p=0.677), suggesting a preserved AC effect in ALL. There was less LT prolongation in ALL-Day7 (134% [123-175%] than in ALL-Day0 (180% [134-238]; p=0.031). This reflects an ongoing dabigatran effect after ASP, albeit to a lesser degree. Conclusions: Our ex-vivo results suggest limited anticoagulant activity of enoxaparin and lack of anticoagulant activity of apixaban and rivaroxaban (both factor Xa inhibitors) in preventing VTE in ALL, in line with prior clinical data. In contrast, dabigatran etexilate, an oral direct thrombin inhibitor not previously tested in ALL, exhibited preserved anticoagulant activity in ALL patients, even after exposure to ASP. This may suggest that dabigatran should be investigated in clinical studies for the unmet need of VTE prophylaxis in ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Children with Acute Lymphoblastic Leukemia and Early Thrombosis Have Dysregulated Coagulation-Related Genes and Pathways

Introduction Thrombo-embolic events (TE) are a frequent complication of childhood acute lymphoblastic leukemia (ALL). Disease-specific mechanistic insights governing the development of TE in this disease remain unclear. Overexpression of coagulation-related genes (coagulome) and pathways have been identified in cancer-induced TE. We investigate whether coagulome dysregulation in newly-diagnosed children with ALL is associated with TE development. Methods We included patients aged 1-21 years old with newly-diagnosed ALL from September 3, 2015, to June 30, 2021, with available RNA-sequencing (RNAseq) data at diagnosis, either enrolled on the Dana-Farber Cancer Institute (DFCI) ALL Consortium Protocol 16-001 or treated following the DFCI 11-001 protocol. RNAseq was performed through an optional study performed as part of the DFCI 16-001 clinical trial for patients who consented to it, or through the research oncogenomic program of the province of Quebec. The primary outcome was the occurrence of TE during ALL therapy requiring medical intervention (grade ≥2 as per the CTCAE version 5). TE were classified as early thrombosis (ET) if they occurred within 6 weeks from treatment start. Patients' characteristics were compared using Chi-Square or Fisher's exact test. Differential gene expression (DE) was performed to compare children with and without TE, adjusted for ALL phenotype (B- or T-cell lineage). The dysregulated genes were compared to a customized panel of 174 genes known to be implicated in cancer-related TE (coagulome and podoplanin) and in coagulation pathways. A secondary analysis, determined a priori, stratified children between ET and no TE, based on the hypothesis that the leukemia environment might predominantly affect early complications. Gene set enrichment analyses (GSEA) were performed on KEGG and gene ontology (GO) databases from the molecular signatures database (MSigDB). DE was considered for absolute fold change (FC) ≥2, and benjamini-Hochberg adjusted p-values (adj p) <0.05. Results We included 274 ALL patients (median age: 6.7 years [interquartile range, IQR: 3.7-12.0 years], 54% male, 81% B-ALL) of whom 49 (17.9%) experienced a TE (including 21 ET) at a median of 76 days (IQR: 27-155 days) following ALL diagnosis. Patients with TE were more likely to be ≥10 years old (57.1% vs. 31.6%, p<0.001) compared to those without TE. Sex, immunophenotype, mediastinal mass, central nervous system disease (CNS) involvement at diagnosis and risk status were otherwise not significantly different among the two groups. Three coagulation pathway-related genes were dysregulated in children with TE compared to those without TE. Glycoprotein 9 (GP9) and platelet factor 4 (PF4), implicated in von Willebrand-dependent platelet adhesion and platelet aggregation, were upregulated (log2FC = 2.1 and 1.5, respectively). Metalloproteinase-2 (MMP2), involved in collagenolysis and thrombus degradation, was downregulated (log2FC = -1.9). The KEGG olfactory transduction pathway was downregulated in the group with TE (normalized enrichment score (NES) = 1.55, adj p<0.001). In the secondary analysis stratified by timing of TE, patients with early TE were more likely to be ≥10 years (80.9% vs. 31.6%, p<0.001), have T-ALL (38.1% vs. 16.9%, p=0.002) and be CNS-positive at diagnosis (52.4% vs. 22.7%, p=0.004). Patients with ET showed overexpression of the GP9, PF4, integrin subunit beta 3 (ITGB3) and clusterin (CLU) genes involved in platelet aggregation and adhesion (log2FC = 3.3, 3.7, 2.7 and 1.6, respectively). The following GO pathways were significantly upregulated: blood coagulation (NES=1.73, adj p<0.001), platelet activation (NES=2.21, adj p<0.01), and platelet alpha granules (NES=2.48, adj p<0.001) while the KEGG olfactory transduction pathway was downregulated (NES=-1.70, adj p<0.001). Dysregulation of the olfactory transduction pathway has previously been described in cancer-associated TE in adults. Conclusion Our study identified coagulation-related genes and pathways that might be drivers of leukemia-induced thrombosis. Early TEs in childhood ALL were associated with significant upregulation of coagulation and platelet activation pathways, suggesting a role of the leukemia microenvironment in TE development. Additional studies are required to validate these observations that could be incorporated in future algorithms to predict TE risk in children with ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pharmacotypes across the Genomic Landscape of Pediatric Acute Lymphoblastic Leukemia and Impact on Treatment Response

INTRODUCTION The current paradigm of risk stratification in childhood acute lymphoblastic leukemia (ALL) utilizes a combination of clinical features, leukemia somatic genomics and early treatment response as measured by minimal residual disease (MRD). Although ALL subtypes and MRD are strongly related to treatment outcomes, the pharmacological basis of their prognostic impact remains incompletely understood. Therefore, we sought to characterize pharmacotypes (drug sensitivities) across the complete spectrum of ALL molecular subtypes and determine their association with in vivo treatment response and survival outcomes. METHODS This was a prospective study of 805 children with newly-diagnosed ALL from Total Therapy XV, XVI, and XVII trials at St Jude Children's Research Hospital. We performed ex vivo pharmacotyping of 18 drugs on primary ALL diagnostic blasts to determine drug sensitivities (LC50 - dose required to kill 50% of leukemia cells). RNA-sequencing was performed to derive molecular subtype. We then evaluated the associations of drug LC50 with clinical features, ALL subtype, MRD, and event-free survival (Figure 1). RESULTS We analyzed 5,447 drug measurements across 23 ALL subtypes. LC50s of prednisolone, asparaginase, and mercaptopurine were associated with age, and/or with leukocyte count at diagnosis. Sensitivities to 14 of 18 drugs varied across ALL molecular subtypes. For cytotoxic agents, B-ALL subtypes with favorable prognosis (ETV6-RUNX1 and hyperdiploidy) exhibited greatest sensitivity to asparaginase and glucocorticoids, whereas high-risk subtypes (KMT2A, BCR-ABL1, and BCR-ABL1-like) were resistant. Novel subtypes DUX4 and ETV6-RUNX1-like ALL showed significant resistance to cytotoxic drugs. For targeted agents, hyperdiploid ALL was most sensitive to venetoclax, whereas TCF3-PBX1, BCR-ABL1, and T-ALL were dasatinib-sensitive. The poor-prognosis ETP-ALL was resistant to most cytotoxic drugs compared to T-ALL. Although BCR-ABL1, BCR-ABL1-like, and CRLF2 are closely related transcriptionally, these three subtypes had distinctive drug sensitivity profiles. In B-ALL, sensitivities of prednisolone, asparaginase, dexamethasone, cytarabine and thiopurines were positively correlated with MRD. By contrast, these drugs were not correlated with MRD in T-ALL. Instead, sensitivities of dasatinib and nelarabine correlated positively, and venetoclax negatively, with MRD in T-ALL. Finally, we classified patients into six functional clusters based on ALL pharmacotypes, each with distinct patterns of somatic genomic aberrations and MRD response. Drug sensitivity cluster was significantly associated with event-free survival, even after adjusting for MRD. Importantly, pharmacotyping identified a T-ALL subset with poor prognosis under conventional chemotherapy but also with unique sensitivity to targeted agents, pointing to therapeutic intervention to improve survival in this group. CONCLUSIONEx vivo drug sensitivities vary widely across ALL subtypes and strongly influence in vivo MRD. Our study comprehensively described pharmacological heterogeneity of ALL and the association of drug sensitivities with survival outcomes, providing insight into the pharmacological basis of inter-patient variability in ALL treatment outcomes and highlighting opportunities for individualizing therapy for this blood cancer. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Observation of Nutritional Status Changes in Patients with Acute Leukemia During Chemotherapy

To assess changes of nutritional status by comprehensive nutrition assessment including nutritional risk screening, dietary assessment, blood biochemical index, and body composition in acute leukemia patients who had undergone chemotherapy. A total of 169 patients with acute leukemia treated at The First Affiliated Hospital of Soochow University from June 2018 to August 2019 were recruited for this study. Before and after chemotherapy, the NRS-2002 and PG-SGA scales, dietary intake, blood biochemical index and body composition were evaluated to compare the changes of nutritional status. NRS-2002 score and PG-SGA score after chemotherapy were significantly increased than those before chemotherapy (P<0.001). Many patients had insufficient nutritional intake during chemotherapy, and the dietary intake score of patients with induction chemotherapy was significantly lower than that of patients with consolidation chemotherapy (P=0.043). The results of multivariate analysis showed that induction chemotherapy was the independent risk factor for the increase of PG-SGA scores and the decrease of dietary intake (all P<0.05). After chemotherapy, the white blood cell count, hemoglobin, and platelet count were significantly decreased (P<0.001), the prealbumin was significantly increased (P<0.001), and the blood glucose was increased (P=0.04), but albumin was not significantly changed. The weight, body mass index, fat-free mass, skeletal muscle mass and intracellular water were all significantly decreased (P<0.001), and visceral fat area was increased significantly after chemotherapy (P<0.05), especially in newly-diagnosed acute lymphoblastic leukemia patients after the induction of chemotherapy. The nutritional status of patients with acute leukemia has undergone significant changes after chemotherapy. A single indicator has limited significance for nutritional status assessment. Comprehensive assessment of nutritional status by multiple tools is worthy of clinical application.

Assessment of Platelet Mitochondrial Respiration in a Pediatric Population: A Pilot Study in Healthy Children and Children with Acute Lymphoblastic Leukemia.

Characterization of mitochondrial respiration in peripheral blood cells has recently emerged as a potential biomarker for the assessment of the severity of hematological malignancies (HM) in adults. Whether changes in platelet respiratory function occur in children with or without HM it is unknown. The present pilot study was double-aimed: (i) to investigate whether platelet respiration is age-dependent in non-HM children and (ii) to assess the platelet mitochondrial respiration in children with newly diagnosed acute lymphoblastic leukemia (ALL). Blood samples obtained from age-grouped children (10–11, 13–14 and 16–17 years) with non-HM and children with ALL (10–11 years) were used to isolate platelets via differential centrifugation. High-resolution respirometry studies of isolated platelets were performed according to a protocol adapted to evaluate complex I and II-supported respiration. An age-related decrease in respiration was observed in the non-HM pediatric population and had comparable values for the 13–14 and 16–17 years. groups. In children with ALL, a significant increase in C I-supported active respiration and decrease in maximal noncoupled respiration were found at the disease onset. In conclusion, in a pediatric population, platelet mitochondrial respiration is age-dependent. Platelet respiratory dysfunction occurs in children with newly-diagnosed ALL, an observation that warrants further investigation of this change as a disease biomarker.

1155. Covering Your Tracts? The Effect of Antibiotic Prophylaxis on Respiratory Tract Infections in Pediatric Acute Lymphoblastic Leukemia

BackgroundAntibiotic prophylaxis decreases rates of febrile neutropenia and systemic infection in children with acute lymphoblastic leukemia (ALL). However, it is unknown whether prophylaxis prevents or ameliorates the severity of specific types of infections like upper respiratory tract infections (URTI) or lower respiratory tract infections (LRTI). MethodsThis is a retrospective, observational convenience cohort study of children with newly-diagnosed ALL, comparing respiratory tract infections (RTI) in participants receiving no antibiotic prophylaxis, levofloxacin prophylaxis, or non-levofloxacin prophylaxis. Information regarding the presence of URTI or LRTI, identified respiratory viruses, hospitalization, oxygen supplementation, and ICU admission was collected through medical record review. The proportion of participants in each group was estimated and compared between groups using Fisher’s exact test and the Kruskal-Wallis test.ResultsOf 262 evaluable participants, 126 received no antibiotic prophylaxis, 59 received levofloxacin prophylaxis, and 77 received non-levofloxacin prophylaxis, with a total of 136 children getting any antibiotic prophylaxis regimen. In the no-prophylaxis group, 22/126 (17.4%) had RTI, compared to 23/136 (16.9%) in the prophylaxis group. There was no significant difference in the numbers of LRTI and URTI, with or without an identified respiratory virus, regardless of the presence or type of antibiotic prophylaxis. Participants receiving prophylaxis did not have a significantly different risk of hospitalization, oxygen supplementation, or ICU admission.Participant Characteristics Comparisons of levofloxacin prophylaxis, other prophylaxis, any prophylaxis, and no prophylaxis ConclusionThere was no observed difference in RTI, hospitalization, oxygen supplementation, or ICU admission for RTI between participants receiving or not receiving antibiotic prophylaxis in this cohort. Because of the relatively low number and severity of respiratory infections, and the high proportion that are viral in etiology, it would likely take a very large sample size to determine the impact of antibacterial prophylaxis on respiratory infections during induction therapy for pediatric ALL.Disclosures Joshua Wolf, MBBS, PhD, FRACP, Karius Inc. (Research Grant or Support) Joshua Wolf, MBBS, PhD, FRACP, Nothing to disclose

Aberrant ARID5B Expression and Its Association with Ikaros Dysfunction in Acute Lymphoblastic Leukemia

Background: AT-rich interactive domain-containing protein 5B (ARID5B) forms a complex with PHD finger protein 2 (PHF2) and the ARID5B-PHF2 complex then induces the demethylation of di-methylated 'Lys-9' of histone H3 (H3K9me2) leading to transcription activation target genes. Mutations and single nucleotide polymorphisms of ARID5B are associated with the development of acute lymphoblastic leukemia (ALL) and treatment outcome. So far there have been no reports about ARID5B expression in ALL and the clinical significance of ARID5BlowPHF2low expression in ALL patients. Ikaros, the product of the IKZF1 gene, plays a critical role in lymphocyte development and functions as a tumor suppressor of leukemia. ChIP-seq data show the obvious Ikaros binding peaks in promoter of ARID5B, which suggested the regulatory role of Ikaros on ARID5B expression in ALL cells. Methods: 164 Subjects with newly-diagnosed ALL (107 B-cell and 57 T-cell ALL; 12-77 years old) between June 2008 and June 2016 were studied at the First Affiliated Hospital of Nanjing Medical University and Zhongda Hospital Southeast University. The study was approved by the Ethics Committee of the institutes. Subjects were allocated in a high or low ARID5B expression cohort (4th quartile vs . 1st-3rd quartiles) with a cut-off value determined by SPSS 20.0. The qChIP assays were performed by incubating chromatin with antibodies against Ikaros, H3K4me3 or normal rabbit IgG as a control. The Nalm6 and CEM cells were transfected with human IKZF1 shRNA or Scrambled shRNA constructs in the GFP vector (pGFP-v-RS) by Neon system. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using uni- and multivariate Cox model. Relapse-free survival (RFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared by log-rank test. Results: We observed the ARID5B mRNA level is significantly down-regulated in subset of ALL patients. Low ARID5B mRNA levels were associated with leukemic cell proliferation and poor prognostic indicators. Particularly ARID5B expression is positively correlated with PHF2 expression in ALL. The ARID5BlowPHF2low was associated with a higher percentage of bone marrow blasts (91.2% vs. 82.4%, P =0.000), stem cell marker CD34+ (88.2% vs. 55.6%, P =0.000), myeloid marker CD33+ (50.9% vs. 28.6%, P =0.036), splenomegaly (50.0% vs. 22.9%, P =0.008), a complete remission(CR) time ≥4 weeks (53% vs. 21%, P =0.003) and higher frequency of Ik 6(+) (49.3% vs. 15.8%, P =0.001) and lower median PLT count (109/L) (32.0 vs. 58.5, P =0.020) compared to patients with non- ARID5BlowPHF2low cohort. The multivariable analysis confirmed the association with bone marrow blasts, Ik 6(+) and a CR time ≥4 weeks. ChIP-seq data shows Ikaros binding peaks in the ARID5B promoter region in Nalm6 B-ALL and primary B-cell ALL cells and the bindings wereconfirmed by individual qChIP assay. Moreover, expression of Ikaros promotes ARID5B expression, while efficient Ikaros knockdown decreased ARID5B expression in Nalm6 and CEM cells. We also found ARID5B expression was significantly lower in B-ALL with Ik6(+) (0.3153 ± 0.0938 vs. 1.2052 ± 0.58441, P =0.02439). Treatment of Nalm6 and CEM cells with CX-4945, which functions as IKAROS activator, promotes expression of ARID5B mRNA in a dose-dependent manner in ALL cell lines and patients' samples, and Ikaros knockdown significantly blocked this effect. Moreover,CX-4945 treatment significantly increases binding of Ikaros and enrichment of H3K4me3 to the ARID5B promoter region in Nalm6 and CEM ALL cells, as well as in patients' samples. Conclusion: We identified that ARID5Blow, particularly ARID5BlowPHF2low expression is associated with leukemia proliferation and poor prognostic markers. Our results also revealed the oncogenic effect of ARID5BlowPHF2low expression in ALL, and identified a high-risk subgroup of ALL characterized by Ikaros dysfunction and ARID5BlowPHF2low expression. DisclosuresDovat:ELF Zone Inc: Membership on an entity's Board of Directors or advisory committees.

Clinical Significance of Phd Finger Protein 2 As a Novel Ikaros Target in Acute Lymphoblastic Leukemia

Background: PHD finger protein 2 (PHF2) mediates demethylation of dimethylated ‘Lys-9‘ of histone H3 (H3K9me2), followed by transcription activation of target genes. PHF2 is a positive epigenetic regulator and is associated with tumor suppression in several types of cancer. Until now, there have been no reports about the PHF2 expression in leukemia patients and its roles in acute lymphoblastic leukemia (ALL).Genetic alterations of IKZF1 encoding the lymphoid transcription factor IKAROS are a hallmark of high risk B-progenitor ALL and are associated with poor outcome. However, it is still undetermined whether and how IKAROS regulates PHF2 expression. Methods: The 164 patients with newly-diagnosed ALL (107 B-ALL and 57 T-ALL; 12-77 years old) were recruited between June 2008 and June 2016 at the First Affiliated Hospital of Nanjing Medical University and Zhongda Hospital Southeast University. The study was approved by the Ethics Committee of the two hospitals. Patients were allocated into high or low PHF2 expression cohorts (4th quartile vs . 1st-3rd quartiles) with a cut-off value determined by SPSS 20.0. The genomic amplification combined direct sequencing was used for detection of IKZF1 deletion. PHF2 expression was determined by qPCR in cells . The enrichment of IKAROS binding and H3K4me3 in the promoter of PHF2 was determined by the chromatin-immunoprecipitation assay combined qPCR (qChIP). IKZF1 knockdown was achieved in cells by transfection of human IKZF1 shRNA constructs in the GFP vector using the Neon Transfection System and with scrambled shRNA as a control. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using univariate and multivariate Cox models. Relapse-free survival and overall survival were estimated by the Kaplan-Meier method and compared by log-rank test. Statistical analyses used SPSS version 20.0. Results: PHF2 mRNA levels in bone marrow samples from adults with ALL, especially those with B-cell ALL (B-ALL), were significantly lower than those in normals. The B-ALL cohort with low PHF2 mRNA levels was associated with a higher median bone marrow blast percentage (90.4% vs. 82.4%; P=0.001), a higher percentage of stem cell marker CD34+ (82.3% vs. 60.0%; P=0.021), myeloid marker CD33+ (48.5% vs. 25.0%; P=0.046), splenomegaly (46.8% vs. 23.1%, P=0.034) and delayed remission more than 4 weeks (48.7% vs. 19.0%; P=0.015), also a higher frequency of Ik6 (+) (46.3% vs. 8.0%, P=0.001) and a lower median PLT count in comparison to the high PHF2 expression cohort. Moreover, ChIP-seq data identified IKAROS binding peaks in the PHF2 promoter region in Nalm6 B-ALL and primary B-ALL cellsand the bindings were confirmed by the qChIP assay . IKAROS overexpression promotes PHF2 mRNA levels, conversely, IKZF1 knockdown decreased PHF2 expression in Nalm6 and CEM cells. These data indicate a direct effect of IKAROS on PHF2 transcription. The patients with IKZF1 deletion (Ik 6) have significantly lower PHF2 expression (0.03201 ± 0.000527 vs. 0.11331± 0.05773; P=0.033), which suggests that the IKZF1 deletion may contribute to low PHF2 expression in ALL patients. Treatment of Nalm6 and CEM cells with CK2 inhibitor CX-4945, which functions as IKAROS activator, promoted expression of PHF2 mRNA in ALL cell lines and primary cells and IKZF1 knockdown blocks this effect. CX-4945 treatment also increases the binding of IKAROS and H3K4me3 enrichment to the PHF2 promoter region in Nalm6 and CEM ALL cells as well as in primary ALL cells. Conclusion: PHF2 low expression was significantly associated with leukemia proliferation and several poor prognostic indicators in adult ALL patients. IKAROS directly regulates PHF2 transcription, and that CK2 inhibitor promotes PHF2 transcription through histone modification in an IKAROS dependent manner. Our data demonstrates the oncogenic effects of IKAROS dysfunction induced decreases in PHF2 expression. This is the first report to demonstrate that low PHF2 expression is significantly associated with Acute Lymphoblastic Leukemia. We suggest that PHF2 expression levelsmay be integrated into future adult ALL risk-stratification models. DisclosuresDovat:ELF Zone Inc: Membership on an entity's Board of Directors or advisory committees.

The Factors Related to Treatment Failure in Children with Acute Lymphoblastic Leukemia

To analyze the efficacy of CCLG-ALL-2008 protocol and the related factors of treatment failure in children with acute lymphoblastic leukemia (ALL). The clinical data of 400 children newly-diagnosed ALL in Children's Hospital of Soochow University from March 1, 2008 to December 31, 2012 was retrospectively analyzed. All the children accepted CCLG-ALL-2008 protocol, and were followed-up until October 2019. The dates of relapse, death and causes of death were recorded. Treatment failure was defined as relapse, non-relapse death, and secondary tumor. Following-up for 10 years, there were 152 cases relapse or non-relapse death, the treatment failure rate was 38%, including 122 relapse (80.3%), 30 non-relapse deaths (19.7%) which included 7 cases (4 cases died of infection and 3 cases died of bleeding) died of treatment (23.3% of non-relapse deaths), 8 cases died of minimal residual disease (MRD) continuous positive (26.7% of non-relapse deaths) and 15 cases died of financial burden (50% of non-relapse deaths). According to the relapse stage, 37 cases (30%) in very early stage, 38 cases (31%) in early stage, and 47 cases (39%) in late stage, while according to the relapse site, 107 cases relapsed in bone marrow, 3 cases in testis, 3 cases in central nervous system (CNS), 5 cases in bone marrow plus testis and 4 cases in bone marrow plus CNS. Bone marrow relapse was the main cause of death in 89 cases, followed by nervous system. Initially diagnosed WBC count (≥50×109/L), T-cell immunophenotype, and MRD-positive at week 12 were the independent risk prognostic factors for relapse in children with ALL, while age (≥10 years), initially diagnosed WBC count (≥50×109/L), M3 bone marrow on day 15, and MRD-positive at week 12 were the independent risk factors due to treatment failure. No secondary tumors were found during the follow-up for 10 years. Relapse is the main cause of treatment failure in children with ALL. The initially diagnosed WBC count, immunophenotype and MRD at week 12 were the independent prognostic factors for relapse of the patients. Financial burden accounts for a large proportion of non-relapse death.

The Dysregulation of Hepcidin-Ferroportin Axis in Childhood Acute Lymphoblastic Leukemia Survivors after Completion of Chemotherapy

Background The hepcidin-ferroportin axis is known to play the essential role on iron homeostasis in human. The binding of hepcidin to ferroportin (FPN) on the membranes of iron-exporting cells induces the ubiquitination of FPN and thereby decreases the delivery of iron to plasma. Low hepcidin with high FPN was well established in patients with ineffective erythropoiesis, e.g. beta-thalassemia major and myelodysplastic syndrome. On the other hand, studies showed increased hepcidin accompanied with decreased FPN in patients with some cancers. Further, anemia in multiple myeloma and Hodgkin's lymphoma were reported to be associated with high hepcidin levels (Blood 2008;112:4292). Here, we investigated the function of the hepcidin-ferroportin axis in childhood acute lymphoblastic leukemia (ALL) survivors who completed chemotherapy. Methods Thirty-three children, aged ≤18 years with ALL and completing TPOG-ALL-2013 protocol before June 30, 2020, were enrolled (ALL-off Gr). According to the treatment protocol, they had been classified as standard-risk (SR) in 20, high-risk (HR) in 12 and very-high-risk (VHR) in 1, respectively. Thirty-eight healthy siblings were assigned as the normal controls (normal Gr) after exclusion of acute illness and iron deficiency. Twenty newly-diagnosed ALL children (ALL-Dx Gr) who did not have sepsis at diagnosis (Dx), and 13 patients with beta-thalassemia major (TM Gr) who received regular transfusion were also enrolled. Serum iron, total iron-binding capacity, ferritin, hepcidin and FPN were collected at Dx in ALL-Dx Gr and at 6-month follow-up after completion of chemotherapy in ALL-off Gr. Selected biochemical assays were checked in normal Gr and TM Gr on the day of routine RBC transfusion. Hepcidin and FPN levels of peripheral blood were measured using ELISA kits (DRG, EIA-5782 for hepcidin &amp; Elabscience, E-EL-H2355 for FPN, respectively). Results The clinical features and biochemical data of all patients are shown in Table 1. The hepcidin and FPN of normal Gr were comparable with those of reference papers. The low hepcidin, high FPN and relatively low hepcidin/ferritin of TM Gr were in consistent with those of published reports. There were no significant differences of age (P =.243) and gender (P =.899) between ALL-off Gr and normal Gr. The ferritin and transferrin saturation between ALL-Dx and ALL-off Grs showed statistical difference (P &amp;lt;.001 and P =.02, respectively). In figure 1, the hepcidin and FPN levels of ALL-Dx Gr were significantly higher than those of the normal Gr (P &amp;lt;.001). The trends remained but did not reach statistical significances between ALL-off and normal Grs. None of ALL-off Gr had mutation of HFE C282Y/H63D, HJV C119F or SLC40A1 C326S. Further, in ALL-off Gr, 6 patients had abnormal ferritin levels (&amp;gt;300 ng/mL; median, 871.55; range, 351.6-959.0)(high-ferritin). MRI scans of liver and heart from the 6 high-ferritin patients did not show any evidence of iron overload. In Figure 2, hepcidin levels were significantly higher in ALL-off patients with high-ferritin or receiving cumulative RBC transfusions &amp;gt;10 units. And ALL-off patients with high-ferritin, RBC transfusions &amp;gt;10 units or receiving HR/VHR treatment regimens had significantly higher FPN levels. Multivariate analysis demonstrated only high-ferritin predicted significantly higher hepcidin (P =.02) and FPN (P &amp;lt;.001) levels in ALL-off Gr. Conclusions The dysregulation of hepcidin-ferroportin axis showed high hepcidin and uncontrolled FPN hyperactivity at Dx of childhood ALL which may point to the impaired down-regulation of FPN by hepcidin. Our findings imply the impaired mechanism may not be fully recovered in ALL survivors after completing chemotherapy, especially those with ferritin &amp;gt;300 ng/mL, HR/VHR groups or receiving higher RBC transfusions. The routine monitoring of the axis function could facilitates the early detection of iron overload in liver and heart. Disclosures No relevant conflicts of interest to declare.

Pediatric-inspired chemotherapy incorporating pegaspargase is safe and results in high rates of minimal residual disease negativity in adults up to age 60 with Philadelphia chromosome-negative acute lymphoblastic leukemia

Administration of pediatric-inspired chemotherapy to adults up to age 60 with acute lymphoblastic leukemia (ALL) is challenging in part due to toxicities of asparaginase as well as myelosuppression. We conducted a multi-center phase II clinical trial (clinicaltrials gov. Identifier: NCT01920737) investigating a pediatric-inspired regimen, based on the augmented arm of the Children’s Cancer Group 1882 protocol, incorporating six doses of pegaspargase 2,000 IU/m2, rationally synchronized to avoid overlapping toxicity with other agents. We treated 39 adults aged 20-60 years (median age 38 years) with newly-diagnosed ALL (n=31) or lymphoblastic lymphoma (n=8). Grade 3-4 hyperbilirubinemia occurred frequently and at higher rates in patients aged 40-60 years (n=18) versus 18-39 years (n=21) (44% vs. 10%, P=0.025). However, eight of nine patients rechallenged with pegaspargase did not experience recurrent grade 3-4 hyperbilirubinemia. Grade 3-4 hypertriglyceridemia and hypofibrinogenemia were common (each 59%). Asparaginase activity at 7 days post-infusion reflected levels associated with adequate asparagine depletion, even among those with antibodies to pegaspargase. Complete response (CR)/CR with incomplete hematologic recovery was observed post-induction in 38 of 39 (97%) patients. Among patients with ALL, rates of minimal residual disease negativity by multi-parameter flow cytometry were 33% and 83% following induction phase I and phase II, respectively. Event-free and overall survival at 3 years (67.8% and 76.4%) compare favorably to outcomes observed in other series. These results demonstrate pegaspargase can be administered in the context of intensive multi-agent chemotherapy to adults aged ≤60 years with manageable toxicity. This regimen may serve as an effective backbone into which novel agents may be incorporated in future frontline studies. Trial registration: https://clinicaltrials. gov/ct2/show/NCT01920737

Capture-based Next-Generation Sequencing Improves the Identification of Immunoglobulin/T-Cell Receptor Clonal Markers and Gene Mutations in Adult Acute Lymphoblastic Leukemia Patients Lacking Molecular Probes.

The monitoring of minimal residual disease (MRD) in Philadelphia-negative acute lymphoblastic leukemia (ALL) requires the identification at diagnosis of immunoglobulin/T-cell receptor (Ig/TCR) rearrangements as clonality markers. Aiming to simplify and possibly improve the patients’ initial screening, we designed a capture-based next-generation sequencing (NGS) panel combining the Ig/TCR rearrangement detection with the profiling of relevant leukemia-related genes. The validation of the assay on well-characterized samples allowed us to identify all the known Ig/TCR rearrangements as well as additional clonalities, including rare rearrangements characterized by uncommon combinations of variable, diversity, and joining (V-D-J) gene segments, oligoclonal rearrangements, and low represented clones. Upon validation, the capture NGS approach allowed us to identify Ig/TCR clonal markers in 87% of a retrospective cohort (MRD-unknown within the Northern Italy Leukemia Group (NILG)-ALL 09/00 clinical trial) and in 83% of newly-diagnosed ALL cases in which conventional method failed, thus proving its prospective applicability. Finally, we identified gene variants in 94.7% of patients analyzed for mutational status with the same implemented capture assay. The prospective application of this technology could simplify clonality assessment and improve standard assay development for leukemia monitoring, as well as provide information about the mutational status of selected leukemia-related genes, potentially representing new prognostic elements, MRD markers, and targets for specific therapies.