Acute Lymphoblastic Leukemia Subtypes Research Articles

Pediatric Preclinical Testing Consortium Evaluation of the MCL1 Inhibitor, AMG 176, Against Xenograft Models of Acute Lymphoblastic Leukemia

Introduction: Children diagnosed with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, however the outcome for high-risk subtypes remains poor. Evasion of apoptosis is a hallmark of cancer, with dysregulation of the BCL-2 family of proteins often reported (Delbridge et al, Nat Rev Cancer 16:99-109, 2016). The BCL-2 family consists of the anti-apoptotic MCL1, BCL-2 and BCLXL as well as pro-apoptotic proteins. AMG 176, a first-in-class MCL1 inhibitor, previously demonstrated potent apoptosis-inducing activity in the nanomolar range against cell lines and patient explants of human hematological malignancies (Caenepeel et al, Cancer Discov 8:1582-97, 2018). Furthermore, discontinuous oral administration of AMG 176 inhibited the growth of human acute myeloid leukemia and multiple myeloma tumor xenografts in vivo at well-tolerated doses. AMG 176 has entered clinical evaluation for patients with hematological malignancies. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium (PPTC) to evaluate the in vivo efficacy of AMG 176 against its patient-derived xenograft (PDX) models of pediatric ALL. Methods:MCL1, BCL-2 and BCLXL mRNA expression was quantified by RNA-seq (http://pedcbioportal.kidsfirstdrc.org). For in vitro cytotoxicity assays, cells were treated with AMG 176 (10 μM - 1 pM) or DMSO control for 24 h and cell viability was determined by AlamarBlue assay and expressed as IC50values. AMG 176 was evaluated in vivo against a panel of 38 ALL PDXs in a single mouse trial format. Each PDX was inoculated via tail vein injection into one NSG mouse (1-5 x 106 cells per mouse). Engraftment and drug responses were determined by enumerating the proportion of human versus mouse CD45+ (%huCD45+) cells in the peripheral blood (PB) at weekly intervals. Treatment with AMG 176 (provided by Amgen, Inc.) began once the %huCD45+ was ≥1% in each mouse and consisted of 60 mg/kg via oral gavage 2 days on, 5 days off for 2 weeks. The experimental endpoint was pre-defined as the %huCD45+ reaching 25% in the PB. Drug efficacy was assessed by event-free survival (EFS) and stringent objective response measures (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). For the pharmacokinetic (PK) study naïve NSG mice were treated with either vehicle or AMG 176 (60 mg/kg) and groups of 3 mice harvested up to 24 h post-treatment. Plasma samples were collected for analysis of AMG 176 using acetonitrile protein precipitation and LC-MS/MS with gradient elution on a Phenomenex Kinetex XB-C18 (3.0 x 50 mm, 2.6 µm) analytical column. Results:MCL1 mRNA expression ranged from 21-282 FPKM across a panel of 90 ALL PDXs representing 6 broad ALL subtypes (B-cell precursor, BCP-ALL; Ph+-ALL; Ph-like ALL; mixed-lineage leukemia-rearranged, MLLr-ALL; T-cell ALL, T-ALL; and early T-cell precursor ALL, ETP-ALL). Thirty eight of the 90 ALL PDXs were selected for in vivo evaluation of AMG 176 activity based on their profiles of MCL1, BCL-2 and BCLXL mRNA expression. AMG 176 was well tolerated in the efficacy study, with only 3 mice experiencing >10% weight loss. Mouse EFS values ranged from 3.5 to 47.8 days from treatment initiation, however no PDX achieved an objective response (remission) with all but one PDX exhibiting progressive disease. The Ph-like ALL PDX, ALL-102 (MCL1 expression 71.8 FPKM) scored a stable disease and had the highest EFS value (47.8 days). In an effort to explain the poor in vivo activity of AMG 176 several follow-up studies were performed. First, the in vitro IC50 values elicited by the AMG-176 stock against 5 leukemia cell lines (MOLM-13, 1.6 µM; NB-4, 2.0 µM; Kasumi-1, 1.6 µM; THP-1, 1.2 µM; and U-937,1.3 µM) were comparable to published data. Second, C18 LC-MS analysis revealed major and minor peaks consistent with theoretical peaks with negligible degradation. Third, PK study results demonstrated that the AMG 176 plasma concentration peaked at 8.5 µM, one hour after administration, with an AUC of 87.7 µM*hr and t1/2 of 4.1 h. This AUC was markedly lower than that in athymic nude mice treated at the same dose (1,120 µM*hr). Conclusions: AMG 176 exhibited modest in vivo activity against a large range of pediatric ALL PDXs, with the best response defined as stable disease in a single PDX. PK data suggest that AMG 176 systemic exposures in NSG mice may not be sufficient to elicit meaningful and prolonged regressions in pediatric ALL PDXs. (Supported by NCI Grants CA199000 and CA199222)

In Silico Analysis of miR-181a and miR-155 and Their Target Genes in the Pathogenesis of Acute Lymphoblastic Leukaemia

Acute lymphoblastic leukaemia (ALL) represents 80% of acute leukaemia in children and 20% in adults. In most cases, the cause remains unknown; however, genetic predisposition is found in most patients. Clinically, a patient is diagnosed with ALL when peripheral blood smear or bone marrow aspirate is overpopulated with lymphoblasts. Although the 5-year overall survival rate exceeds 90% using available treatments, the life-threatening effects still affect some patients [1]. Therefore, further improvement in clinical outcomes and reduction of adverse effects require novel therapeutic approaches such as microRNAs (miRNAs) biomarkers. In addition, miRNAs have demonstrated distinct expression profiles in classifying the ALL subtypes and involved in various cancers [2].
 
 Previously, miR-181a and miR-155 have been discussed, either acting as oncogenic or tumour suppressive miRNAs in carcinogenesis. The study aims to investigate the role of miR-181a and miR-155 and their predicted target genes in the pathogenesis of ALL. Hence, in silico analysis was conducted using miRDB 6.0 software to identify the predicted target genes and GeneCodis 4.0 software to identify signalling pathways regulated by miR-181a and miR-155. As presented in Table 1, miR-181a was found to be downregulated whereas miR-155 was significantly upregulated in ALL groups as compared to control groups [3].
 Analysis of target expression was conducted to identify predicted target genes in ALL cell line, DND-41. In this study, the predicted target genes are regulated by miR-181a and miR-155 (with target score ≥ 80 and target expression ≥ 20) revealed 57 genes and 29 genes, respectively. The most significant genes regulated by miR-181a and miR- 155 were DUSP6 and H3F3A, respectively. DUSP6 is a regulator for hyperactivation of the extracellular signal- regulated kinase (ERK) in relapsed pre-B ALL patients. Thus, this reaction caused lesions in the RAS pathway and oncogenic tyrosine kinases (e.g., BCR-ABL1).
 
 Additionally, a complete transformation of pre-B ALL clones was done by BCR-ABL1 and correlated with the activity of other MAPKs such as MAPK14 and MAP2K1. Therefore, ERK was identified as a negative feedback regulator of the RAS-ERK pathway [4].
 
 H3F3A was reported to be involved in the pathogenesis of paediatric leukaemia caused by mutated genes that encoded proteins during epigenetic regulation. The mutation in H3F3A led to the inability of this residue to undergo periodic regulatory post-translational modifications and blocked trimethylation of all H3 during T-ALL pathogenesis. Specifically, the recurrent T-ALL patients found the lysine-to-arginine mutations at H3K27 and H3K36. Hence, the reduced expression of H3 proteins was associated with unfavourable prognosis [5].
 
 The functional enrichment analysis of KEGG pathway was conducted by using GeneCodis 4.0 software. The most significant pathway targeted by miR-181a and miR-155 was p53 signalling pathway and MAPK pathway, respectively.
 
 SESN3 was mainly responsive to p53 protein by inducing its expression upon oxidative stress and strictly associated with the antioxidant response during p53 signalling pathway. Hence, SESN3 could be a potential therapeutic drug for anticancer therapy, such as decreased mTORC1 mechanism in leukaemia patients [6]. Besides that, FOS was disrupted the MAPK pathway components, specifically MEK1 and MEK2 which could increase sensitivity to chemotherapy in relapsed ALL patients. These components were involved in ERK phosphorylation that critically drives chemoresistance during MAPK/ERK signalling pathway [7].
 
 In conclusion, differentially expressed miRNAs have promising roles and can be used as effective biomarkers for diagnostic, prognostic, predictive and therapeutic effects in ALL patients.

The role of E3 ubiquitin ligase WWP2 and the regulation of PARP1 by ubiquitinated degradation in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) has been a huge threat for people's health and finding effective target therapy is urgent and important. WWP2, as one of E3 ubiquitin ligase, is involved in many biological processes by specifically binding to substrates. PARP1 plays a role in cell apoptosis and is considered as a therapeutic target of certain cancers. In this study, we firstly found that WWP2 expressed higher in newly diagnosed ALL patients comparing with complete remission (CR) ALL patients and normal control people, and WWP2 in relapse ALL patients expressed higher than normal control people. WWP2 expression was related with the FAB subtype of ALL and the proportion of blast cells in bone marrow blood tested by flow cytometry. We demonstrated knockout WWP2 inhibited the ALL growth and enhanced apoptosis induced by Dox in vitro and vivo for the first time. WWP2 negatively regulated and interacted with PARP1 and WWP2 mechanically degraded PARP1 through polyubiquitin-proteasome pathway in ALL. These findings suggested WWP2 played a role in ALL development as well as growth and apoptosis, and also displayed a regulatory pathway of PARP1, which provided a new potential therapeutic target for the treatment of ALL.

Combination of Cross Stage Partial Network and GhostNet with Spatial Pyramid Pooling on Yolov4 for Detection of Acute Lymphoblastic Leukemia Subtypes in Multi-Cell Blood Microscopic Image

Purpose: Acute Lymphoblastic Leukemia (ALL) Detection with microscopic blood images can use a deep learning-based object detection model to localize and classify ALL cell subtypes. Previous studies only performed single cell-based detection objects or binary classification with leukemia and normal classes. Detection of ALL subtypes is crucial to support early diagnosis and treatment. Therefore, an object detection model is needed to detect ALL subtypes in multi-cell blood microscopic images.Methods: This study focuses on detecting the ALL subtype using YOLOV4 with a modified neck using Cross Stage Partial Network (CSPNet) and GhostNet. CSPNet is combined with Spatial Pyramid Pooling (SPP) to become SPPCSP to get various features map before the YOLOv4 final layer. Ghostnet was used to reduce the computation time of the modified YOLOV4 neck.Result: Experimental results show that YOLOv4 SPPCSP outperformed the recall value of 14.6%, the value of mAP@.5 0.8%, and reduced the computation time by 4.7 ms compared to the original YOLOv4.Novelty: The combination of CSPNet and GhostNet for YOLOV4 neck modification can increase the variety of features map and reduce computing time compared to the Original YOLOv4.

ALL-350 Characterization of Acute Lymphoblastic Leukemia in the Pediatric Mexican Mestizo Population

Acute lymphoblastic leukemia (ALL) is the most common neoplasm of lymphoid progenitor cells in pediatric populations. ALL cases can be broadly classified as B-cell or T-cell. B-cell ALL (B-ALL) has many genetic subtypes characterized by major chromosomal alterations. Flow cytometry and biomarkers identification provide information about the subtype of ALL and can be correlated with the patient's prognosis. Based on reactivity with antigens present on the surface or in the cytoplasm of leukemia cells, B-ALL can be divided into 4 subtypes according to its stage of differentiation: pro-B, common B, pre-B, and immature B subtypes. This study aims to establish the association of the molecular biomarkers TCF3-PBX1 t(1;19), rearrangement of MLL t(4;11), BCR-ABL1 t(9;22), and ETV6-RUNX1 t(12;21) with ALL subtypes according to its stage of differentiation. The study includes a retrospective analysis of laboratory data results from the Mexican mestizo pediatric population diagnosed with ALL during a 3-year period (2018-2021), referred to Mendel laboratories from different health institutions. Ninety ALL cases, age range 0-17 years, were classified by immunophenotype and tested by PCR for the biomarkers TCF3-PBX1, rearrangement of MLL, BCR-ABL1, and ETV6-RUNX1. The biomarkers tested were expected to be present in most of the cases (>50%) regardless of the differentiated ALL subtype. B-cell ALL represented 92.2% of the cases (83); the prevailing subtype was pre-B with 51.1% of all cases. The biomarkers TCF3-PBX1, rearrangement of MLL, BCR-ABL1, and ETV6-RUNX1 were present in at least one case in the subtype pro-B. ETV6-RUNX1 was identified as the only marker in common B-ALL. Seventy-one percent of the cases showed no presence of the 4 biomarkers tested and correspond to quadruple negative cases. The biomarkers TCF3-PBX1, rearrangement of MLL, BCR-ABL1, and ETV6-RUNX1 are decisive in establishing the prognosis of patients with any of the ALL-B subtypes and enable the definition of a stratified treatment. The absence of these markers prevails in the study population, so it is essential to include other recently described mutations in laboratory determinations. Laboratorios MENDEL, Morelia Michoacán. México.

RISK FACTORS FOR ACUTE KIDNEY INJURY IN OUTPATIENT ADMINISTRATION OF HIGH-DOSE METHOTREXATE IN ADULTS WITHOUT DRUG MONITORING

Methotrexate is an antifolate that has been an essential part of chemotherapy regimens for acute lymphoblastic leukemia (ALL) and specific subtypes of non-Hodgkin lymphoma (NHL), when it is used in high-dose (HD, ≥ 1 g/m2) to overcome the blood-brain barrier. Nephrotoxicity is the major toxicity of HDMTX, possibly inducing critical organ damage and death if not early treated. Although some factors for drug-induced acute kidney injury (AKI) have been pointed out, the reality is that in resource-poor settings, HDMTX is usually administered in an outpatient clinic and without drug monitoring. The exact incidence of AKI after MTX in these conditions was not reported in adults yet. In this study, we intend to summarize our toxicity data after outpatient administration of HDMTX without drug monitoring. We aim to find risk factors and describe their outcomes in a real-life setting. This is a retrospective nested case-control study conducted at a large academic medical center in Sao Paulo, Brazil. Patients from 16 years old and above with ALL and NHL who received at least one outpatient infusion of HDMTX without drug level monitoring between Jan/2010 and Jun/2020 were included. Cases were those patients who developed AKI by KDIGO definition or with delayed MTX clearance (serum MTX level > 0.1 mM after 72h from drug infusion), when clinically suspected. A nested cohort of controls was composed of patients who did not develop the complication, sampling from a larger cohort of patients who received HDMTX. Cases and controls were randomly matched 1:2 for the employed protocol only. Conditional logistic regression was used to find baseline risk factors. A total of 404 patients received at least one HDMTX, with 102 not fulfilling the inclusion criteria. Among 302 patients included, 840 infusions were performed (median 2 infusions/patient, range 1-10). Most patients had a diffuse large B-cell lymphoma (DLBCL) (37%), Burkitt lymphoma (BL) (19.5%), or ALL (18%) diagnosis. Most subjects received HDMTX within a course of Hyper-CVAD (HCVAD) or monotherapy (61%). Hospitalization occurred in 8.6% of infusions during the MTX nadir, with 4.6% needing intensive care, being febrile neutropenia the most common complication (63%). Twenty-five patients presented AKI after HDMTX administration, corresponding to 3% (95% CI 2-4.4) of MTX infusions and 8.3% (95% CI 5.5-12.1) of patients. Univariate analysis for finding pre-treatment factors related to AKI after HDMTX were age>44 y (OR 3.2 [95% CI 1 - 9.7], p = 0.045), body surface≥1.76 m2 (OR 3.4 [95% CI 1.2-9.9], p = 0.039), BMI≥23.8 kg/m2 (OR 3.5 [95% CI 1.2-9.9], p = 0.02), baseline creatinine clearance (OR 0.96 [95% CI 0.93-0.99], p = 0.012), thrombocytopenia (<150x109/L) (OR 4.7 [95% CI 1.7-13.1], p = 0.002). Multivariate analysis for adjusting such factors found that only BMI is independently related to AKI after HDMTX in our setting (OR 3.8 [95% CI 1.2-11.8], p = 0.06). Among these cases, 9/25 had their MTX serum level measured at the AKI, with delayed MTX clearance in 7/9. HIV-associated BL was more frequent in cases (18 vs. 6.8%, p = 0.03). 22/25 needed hospitalization, with infection diagnosed in 23/25 e need for dialysis in 5/25 cases. Death after AKI occurred in 56% (14/25). Our data showed a similar rate of AKI after HDMTX to that reported in the literature, even without drug monitoring. However, patients who developed AKI in our cohort fare worse than expected, with more hospitalizations and death. Higher BMI was associated with MTX-induced AKI in our cohort, suggesting a differential drug clearance and the need for specific guidelines for obese patients.

Exploring blue spaces’ effects on childhood leukaemia inci-dence: A population-based case-control study in Spain

Background and aim: Blue spaces have been a key part of human evolution, providing resources and helping economies develop. To date, no studies have been carried out to explore how they may be linked to paediatric oncological diseases. The aim of this study was to explore the possible relationship of residential proximity to natural and urban blue spaces on childhood leukaemia. Methods: A population-based case control study were conducted in four regions of Spain across the period 2000-2018. 936 incident cases and 5,616 controls were included, individually matched by sex, year-of-birth and place-of-residence. An exposure proxy with four distances (250m, 500m, 750m and 1 km) to blue spaces was built using the geographical coordinates of the participants’ home residences. Odds ratios (ORs) and 95% confidence intervals (95%CIs), blue space exposure were calculated for overall childhood leukaemia, and the acute lymphoblastic (ALL) and acute myeloblastic leukaemia (AML) subtypes, with adjustment for socio-demographic and environmental covariates. Results: A decrease in overall childhood leukaemia and ALL-subtype incidence was found as we came nearer to children’s places of residence, showing, for the study as a whole, a reduced incidence at 250m (odds ratio (OR)=0.77; 95%CI=0.60-0.97), 500m (OR=0.78; 95%CI=0.65-0.93), 750m (OR=0.80; 95%CI=0.69-0.93) and 1000m (OR=0.84; 95%CI=0.72-0.97). AML model results showed an increasing incidence at closest to subjects’ homes (OR at 250m = 1.06; 95%CI=0.63-1.71). Conclusions: Our results suggest a possible association between lower childhood-leukaemia incidence and blue-space proximity. This study is a first approach to blue spaces’ possible effects on childhood leukaemia incidence; consequently, it is necessary to continue studying these spaces – while taking into account more individualized data and other possible environmental risk factors. Keywords: urban blue spaces; environmental factors; childhood cancer; childhood leukaemia; incidence; spatial epidemiology.

Prospective longitudinal evaluation of treatment-related toxicity and health-related quality of life during the first year of treatment for pediatric acute lymphoblastic leukemia

BackgroundPediatric acute lymphoblastic leukemia (ALL) therapy is accompanied by treatment-related toxicities (TRTs) and impaired quality of life. In Australia and New Zealand, children with ALL are treated with either Children’s Oncology Group (COG) or international Berlin-Frankfurt-Munster (iBFM) Study Group-based therapy. We conducted a prospective registry study to document symptomatic TRTs (venous thrombosis, neurotoxicity, pancreatitis and bone toxicity), compare TRT outcomes to retrospective TRT data, and measure the impact of TRTs on children’s general and cancer-related health-related quality of life (HRQoL) and parents’ emotional well-being.MethodsParents of children with newly diagnosed ALL were invited to participate in the ASSET (Acute Lymphoblastic Leukaemia Subtypes and Side Effects from Treatment) study and a prospective, longitudinal HRQoL study. TRTs were reported prospectively and families completed questionnaires for general (Healthy Utility Index Mark 3) and cancer specific (Pediatric Quality of Life Inventory (PedsQL)-Cancer Module) health related quality of life as well the Emotion Thermometer to assess emotional well-being.ResultsBeginning in 2016, 260 pediatric patients with ALL were enrolled on the TRT registry with a median age at diagnosis of 59 months (range 1–213 months), 144 males (55.4%), majority with Pre-B cell immunophenotype, n = 226 (86.9%), 173 patients (66.5%) treated according to COG platform with relatively equal distribution across risk classification sub-groups. From 2018, 79 families participated in the HRQoL study through the first year of treatment. There were 74 TRT recorded, reflecting a 28.5% risk of developing a TRT. Individual TRT incidence was consistent with previous studies, being 7.7% for symptomatic VTE, 11.9% neurotoxicity, 5.4% bone toxicity and 5.0% pancreatitis. Children’s HRQoL was significantly lower than population norms throughout the first year of treatment. An improvement in general HRQoL, measured by the HUI3, contrasted with the lack of improvement in cancer-related HRQoL measured by the PedsQL Cancer Module over the first 12 months. There were no persisting differences in the HRQoL impact of COG compared to iBFM therapy.ConclusionsIt is feasible to prospectively monitor TRT incidence and longitudinal HRQoL impacts during ALL therapy. Early phases of ALL therapy, regardless of treatment platform, result in prolonged reductions in cancer-related HRQoL.

Prognostic factors and outcomes of infant acute lymphoblastic leukemia (ALL), hypodiploid ALL, and mixed-phenotype acute leukemia (MPAL) in Canada: a report from CYP-C

The epidemiology of infant acute lymphoblastic leukemia (ALL), hypodiploid ALL, and mixed-phenotype acute leukemia (MPAL) in Canada is unknown. The main objective was to describe the prevalence, prognostic factors, and outcomes of three rare and high-risk ALL subtypes in Canada. This is a retrospective study using the Cancer in Young People-Canada (CYP-C) database. Event-free survival (EFS) and overall survival (OS) were described by the Kaplan–Meier method and compared using the log-rank test. Among 2626 children aged 0–14 years diagnosed with B-cell acute lymphoblastic leukemia (B-ALL) between 2001 and 2018, 227 (8.6%) patients were identified to be infant ALL (n = 139), hypodiploid ALL (n = 43), or MPAL (n = 45). The 5-year EFS/OS was significantly worse in the infant ALL subgroup compared to that of hypodiploid ALL and MPAL. For the entire cohort, presenting White blood cells (WBCs) ≥50 × 109/L was independently associated with worse EFS/OS.

The genomic landscape of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Here, using whole-genome, exome and transcriptome sequencing of 2,754 childhood patients with ALL, we find that, despite a generally low mutation burden, ALL cases harbor a median of four putative somatic driver alterations per sample, with 376 putative driver genes identified varying in prevalence across ALL subtypes. Most samples harbor at least one rare gene alteration, including 70 putative cancer driver genes associated with ubiquitination, SUMOylation, noncoding transcripts and other functions. In hyperdiploid B-ALL, chromosomal gains are acquired early and synchronously before ultraviolet-induced mutation. By contrast, ultraviolet-induced mutations precede chromosomal gains in B-ALL cases with intrachromosomal amplification of chromosome 21. We also demonstrate the prognostic significance of genetic alterations within subtypes. Intriguingly, DUX4- and KMT2A-rearranged subtypes separate into CEBPA/FLT3- or NFATC4-expressing subgroups with potential clinical implications. Together, these results deepen understanding of the ALL genomic landscape and associated outcomes.

Characteristics of patients with acute lymphoblastic leukemia in 2016-2020 at Sanglah General Hospital, Bali

Background: Acute lymphoblastic leukemia (ALL) is a bone marrow malignancy that is the most common cause of death in children. This study aims to determine the characteristics of ALL patients in 2016-2020 at Sanglah Hospital Denpasar, Bali.&#x0D; Methods: This study used a retrospective descriptive study with secondary data from 166 patients. Variables assessed in this study included: demographic characteristics (age, gender, place of origin), complete blood count results include of hemoglobin levels, leukocyte (white blood cell) count, absolute neutrophil count, absolute lymphocyte count, thrombocyte count, and morphology subtype of ALL based on FAB classification from bone marrow aspiration (BMA).&#x0D; Results: From demographic characteristics: most patients' age was in school-age children (&gt;6 - &lt;18 years) about 59.7%, dominated by male (60.2%), place of origin from Bali (71.7%) which is from Denpasar city (23.5%). From the laboratory results, complete blood count found that the most results are moderate anemia in children (54.7%), in adult female (70%), and in adult male (52.9%); leukocytosis in children (41%), in adult (77.8%); neutropenia in children (61.2%), in adult (59.3%); lymphocytosis in children (43.2%), in adult (81.5%); thrombocytopenia in children (97.1%), in adult (100%). From the results of the BMA examination, based on the FAB (French, American, and British) classification, the most common morphology of ALL was L2 subtype (78.9%).&#x0D; Conclusion: Characteristics of patients with ALL at Sanglah General Hospital are dominated by children, male, from Denpasar, Bali, with laboratory results showing moderate anemia, leukocytosis, neutropenia, lymphocytosis, thrombocytopenia, and the most common morphological variants are ALL-L2 subtype.

Commonly Assessed Markers in Childhood BCP-ALL Diagnostic Panels and Their Association with Genetic Aberrations and Outcome Prediction.

Immunophenotypic characterization of leukemic cells with the use of flow cytometry (FC) is a fundamental tool in acute lymphoblastic leukemia (ALL) diagnostics. A variety of genetic aberrations underlie specific B-cell precursor ALL (BCP-ALL) subtypes and their identification is of great importance for risk group stratification. These aberrations include: ETV6::RUNX1 fusion gene, Philadelphia chromosome (BCR::ABL1 fusion gene), rearrangements of the KMT2A, TCF3::PBX1 fusion gene and changes in chromosome number (hyperdiploidy and hypodiploidy). Diagnostic panels for BCP-ALL usually include B-cell lineage specific antigens: CD19, CD10, CD20, maturation stage markers: CD34, CD10, CD38, TdT, IgM and other markers useful for possible genetic subtype indication. Some genetic features of leukemic cells (blasts) are associated with expression of certain antigens. This review comprehensively summarizes all known research data on genotype-immunophenotype correlations in BCP-ALL. In some cases, single molecules are predictive of particular genetic subtypes, i.e., NG2 with KMT2A gene rearrangements or CD123 with hyperdiploidy. However, much more information on possible genotype or prognosis can be obtained with wider (≥8-color) panels. In several studies, a quantitative antigen expression scale and advanced statistical analyses were used to further increase the specificity and sensitivity of genotype/immunophenotype correlation detection. Fast detection of possible genotype/immunophenotype correlations makes multicolor flow cytometry an essential tool for initial leukemia diagnostics and stratification.

Epigenetic regulator genes direct lineage switching in MLL/AF4 leukemia

AbstractThe fusion gene MLL/AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukemia. Relapse can be associated with a lineage switch from acute lymphoblastic to acute myeloid leukemia, resulting in poor clinical outcomes caused by resistance to chemotherapies and immunotherapies. In this study, the myeloid relapses shared oncogene fusion breakpoints with their matched lymphoid presentations and originated from various differentiation stages from immature progenitors through to committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programs, including alternative splicing. These findings indicate that the execution and maintenance of lymphoid lineage differentiation is impaired. The relapsed myeloid phenotype is recurrently associated with the altered expression, splicing, or mutation of chromatin modifiers, including CHD4 coding for the ATPase/helicase of the nucleosome remodelling and deacetylation complex. Perturbation of CHD4 alone or in combination with other mutated epigenetic modifiers induces myeloid gene expression in MLL/AF4+ cell models, indicating that lineage switching in MLL/AF4 leukemia is driven and maintained by disrupted epigenetic regulation.

Association of TNF-α rs1800629 with Adult Acute B-Cell Lymphoblastic Leukemia.

TNF–α influences lymphomagenesis by upregulating proinflammatory and antiapoptotic pathways. In this study, we evaluated the frequency of TNF–α rs1800629 (–308 G>A) polymorphism in newly diagnosed adult patients with acute lymphoblastic leukemia (ALL) and its correlation with age at diagnosis, gender and subtype of ALL. In this case control study, a total of 330 individuals were recruited, including 165 newly diagnosed adult patients with ALL, from the Radiation and Isotope Center in Khartoum (RICK) and 165 healthy normal controls. TNF–α rs1800629 polymorphism was tested through allele-specific polymerase chain reaction (PCR) assay. The frequency of the rs1800629 GA genotype was high (70.9% vs. 60%, OR = 1.84) in the patient group as compared to healthy controls, whereas GG and AA genotypes did not exhibit any statistically significant difference between controls and patients. Based on subtype, GG and GA rs1800629 genotypes showed increased risk of B-ALL (OR 0.46 and 2.12, respectively), whereas rs1800629 GG, GA and AA genotypes did not show any disease association with T-ALL (p > 0.05). Age at diagnosis and gender did not exhibit any association of rs1800629 with ALL in the patient group. In conclusion, rs1800629 is associated with high risk of adult B-ALL, with an insignificant effect of age at diagnosis and gender.

JAK2 Alterations in Acute Lymphoblastic Leukemia: Molecular Insights for Superior Precision Medicine Strategies.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, arising from immature lymphocytes that show uncontrolled proliferation and arrested differentiation. Genomic alterations affecting Janus kinase 2 (JAK2) correlate with some of the poorest outcomes within the Philadelphia-like subtype of ALL. Given the success of kinase inhibitors in the treatment of chronic myeloid leukemia, the discovery of activating JAK2 point mutations and JAK2 fusion genes in ALL, was a breakthrough for potential targeted therapies. However, the molecular mechanisms by which these alterations activate JAK2 and promote downstream signaling is poorly understood. Furthermore, as clinical data regarding the limitations of approved JAK inhibitors in myeloproliferative disorders matures, there is a growing awareness of the need for alternative precision medicine approaches for specific JAK2 lesions. This review focuses on the molecular mechanisms behind ALL-associated JAK2 mutations and JAK2 fusion genes, known and potential causes of JAK-inhibitor resistance, and how JAK2 alterations could be targeted using alternative and novel rationally designed therapies to guide precision medicine approaches for these high-risk subtypes of ALL.

Innovative Approaches to the Management of Acute Lymphoblastic Leukemia Across the Age Spectrum.

Adults compose nearly half of all patients diagnosed with acute lymphoblastic leukemia (ALL) and historically have had poor survival compared with pediatric patients. Recently approved therapies, such as monoclonal antibodies, CAR T-cell constructs, and next-generation tyrosine kinase inhibitors, have improved survival in relapsed and refractory ALL, and studies are now examining incorporating these treatments and others into the upfront setting. In adolescent and young adult patients, use of pediatric-based regimens has already improved survival compared with historical controls, and the addition of monoclonal antibodies, such as inotuzumab ozogamicin and blinatumomab, may further enhance this survival benefit. In older adults, approaches have centered on minimizing conventional chemotherapy to decrease toxicity by incorporating monoclonal antibodies and other novel therapies to increase efficacy. With the addition of tyrosine kinase inhibitors to chemotherapy for patients with Philadelphia chromosome-positive ALL, survival of this once poor-prognosis ALL subtype now approaches or exceeds outcomes of other subtypes of adult ALL. Further refinements in the backbone treatment regimen and optimal consolidation approaches will likely improve survival further. Although allogeneic hematopoietic stem cell transplant was previously routinely used as consolidation for adults with ALL, incorporation of measurable residual disease and other risk stratification strategies has enabled better identification of patients who will benefit from allogeneic hematopoietic stem cell transplant. Ongoing clinical trials investigating these approaches will continue the evolution of treatment approaches for adults with ALL, with further improvement in outcomes anticipated.

P333: MULTI-COHORT GENE EXPRESSION RESOURCE FOR AUTOMATIC SUBTYPING OF B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and comprises multiple distinguishable genomic subtypes. RNA sequencing provides a functional snapshot that we propose will provide rapid diagnostic information for most ALL subtypes and deep biological insight to guide diagnostic efforts of atypical cases. Aims: The aims of this study were to elucidate the predictive associations between mRNA-seq profiles and independently verified genomic lesions, and to develop easy-to-interpret transcriptome-wide biomarkers for ALL subtyping in the clinical setting. Methods: A total of 1,279 ALL patients were included in the discovery dataset from six publicly available North American cohorts and 767 Australian ALL patients were included as an external validation cohort. Additional publicly available mRNA-seq data (n = 1,160) were used for quality control modelling. A novel batch correction method was introduced and applied to adjust for cohort differences. Three machine learning models (random forest, projections to latent structures and nearest Euclidean neighbor) were trained with the discovery set and validated in the Australian dataset. Results: Out of 18,503 genes with usable expression, 11,830 (64%) were confounded by cohort effects and excluded. There were no substantial differences between the machine learning methods in the validation set; we chose the nearest neighbor technique as the simplest alternative. Six ALL subtypes (ETV6-RUNX1, KMT2A, DUX4, PAX5 P80R, TCF3-PBX1, ZNF384) that covered 32% of patients were robustly detected by mRNA-seq (PPV ≥ 87%). Five other frequent subtypes (CRLF2, hypodiploid, hyperdiploid, PAX5 alterations and Ph-positive) were distinguishable in 40% of patients, although overlapping transcriptional profiles led to lower accuracy (52% ≤ PPV ≤ 73%). Based on these findings, we developed the Allspice R package that predicts ALL subtypes and driver genes from unadjusted mRNA-seq read counts as encountered in real-world settings. Image:Summary/Conclusion: We demonstrated high diagnostic accuracy for a fully automated mRNA-seq method for a third of ALL patients and additional contribution to diagnostic efforts for patients with inconclusive or mixed genomic characteristics.

Immunophenotypic characteristics of ZNF384 rearrangement compared with BCR-ABL1, KMT2A rearrangement, and other adult B-cell precursor acute lymphoblastic leukemia.

ZNF384 rearrangement has been recently identified as a new subtype of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, comprehensive studies clarifying immunophenotypic features and discriminating them from non-ZNF384 in adult BCP-ALL remain scarce to date. Flow cytometric assessments were retrospectively performed in 43 patients with ZNF384 rearrangement, 45 with BCR-ABL1, 29 with KMT2A rearrangement and 44 with other BCP-ALL in the analysis cohort. CD33- and CD13-positive frequencies were significantly higher in patients with ZNF384 rearrangement than in those with non-ZNF384; however, no significant difference was observed in CD10- and CD123-positive frequencies. Analysis of antigen-positive cell proportion and median fluorescence intensity (MFI) further indicated that patients with ZNF384 rearrangement had significantly lower CD10 and higher CD33, CD13, and CD123 proportion and MFI. However, compared with KMT2A rearrangement, the CD10 expression in patients with ZNF384 rearrangement was higher, with the median percentage and MFI of 36.16 (3.63-94.79)% versus 4.53 (0.03-21.00)%, and 4.50 (0.86-32.26) versus 2.06 (0.87-4.04), respectively (p < 0.0001). Furthermore, compared with BCR-ABL1 and other BCP-ALL, ZNF384 rearrangement had significantly higher CD33 and CD13 proportion and MFI (p < 0.0001 and p < 0.05, respectively). In addition, higher CD123 proportion and MFI in ZNF384 rearrangement than those in the other three groups were reported for the first time (p < 0.01). A flow cytometry scoring system, including CD10%, CD33MFI, CD13%, and CD123MFI, was proposed and verified to predict ZNF384 rearrangement with high sensitivity and specificity, that is, 76.74% and 91.53% in the analysis and 87.50% and 91.30% in the validation cohort. The multiparameter immunophenotypic scoring system could suggest ZNF384 rearrangement.

Abstract CT027: Exploration of association of leukemic molecular profile with efficacy in patients (pts) with relapsed/refractory acute lymphoblastic leukemia (ALL) treated with inotuzumab ozogamicin (InO) in the phase 3 INO-VATE trial

Abstract Background: INO-VATE (NCT01564784) demonstrated higher rates of remission, MRD-negativity, and improved overall survival (hazard ratio 0.75, one-sided P=0.01) for pts with R/R ALL who received InO vs standard chemotherapy (SC). Here we explore the potential association between leukemic molecular profiles and efficacy of InO. Methods: Pts with CD22+ ALL due to receive salvage treatment were randomized to InO or SC; clinical methods and results were published previously (Kantarjian HM, et al. N Engl J Med 2016; PMID: 27292104). Bone marrow samples collected at screening post protocol amendment 2 underwent RNA-sequencing with analysis of gene expression profile, fusions, and genomic alterations. Data cutoff: Jan 4, 2017. Results: Of 326 pts in the INO-VATE intent-to-treat (ITT) population, 91 (InO, 43; SC, 48) had samples evaluable for molecular profiling. Baseline characteristics were similar in the evaluable pts vs the ITT population. Most common (≥5%) ALL subtypes were BCR-ABL1-positive (14%), low hypodiploidy (12%), Philadelphia chromosome (Ph)-like (11%), KMT2A-rearranged (9%), and DUX4-rearranged (6%); 29% (26/91) of pts could not be subtyped due to low blast counts. Most common (≥5%) non-BCR-ABL1 oncogenic fusions detected were IGH-CRLF2 (8%), and KMT2A-AFF1 (6%). Among non-fusion genomic alterations, those involving chromatin modifying genes featured prominently (e.g. KMT2D, 18%; CREBBP, 12%; KMT2C, 11%; ARID2, 9%; ARID1A, 8%; SET2D, 7%; EP300, 6%). Other commonly altered (≥9%) genes included TP53 (14%), NOTCH1 (11%), KRAS (9%), and PAX5 (9%). Similar to the overall findings of INO-VATE, complete remission (CR)/CR with incomplete count recovery (CRi) rates were numerically higher with InO vs SC across ALL subtypes, including but not limited to, low hypodiploidy (67% [2/3] vs 13% [1/8], P=0.15), Ph-like (83% [5/6] vs 0% [0/4], P=0.02), and KMT2A (100% [2/2] vs 17% [1/6], P=0.11), although not in BCR-ABL1-positive (67% [4/6] vs 57% [4/7], P=0.59). Within pts bearing common non-BCR-ABL1 oncogenic fusions, CR/CRi was seen for InO including for IGH-CRLF2 (50% [1/2] vs 20% [1/5] for SC), KMT2A-AFF1 (100% [1/1] vs 25% [1/4] for SC), and IGH-DUX4 (100% [3/3] vs 0% [0/1] for SC). Consistent with a previous report (Yang X, et al. Blood 2019; 134 [Suppl 1]: 3888) CR/CRi rates were high for InO in pts with TP53 alterations (100% [5/5] vs 13% [1/8] for SC, P=0.0047). Conclusions: This retrospective, exploratory analysis of INO-VATE demonstrated potential for benefit with InO for ALL pts across leukemic subtypes, including Ph-like, and for those bearing diverse fusions. Further exploration of Ph-like subtype or TP53 alterations as candidate predictive biomarkers for InO in pts with R/R ALL is warranted. Citation Format: Yaqi Zhao, A Douglas Laird, Kathryn Roberts, Rolla Yafawi, Hagop Kantarjian, Daniel J. DeAngelo, Matthias Stelljes, Michaela Liedtke, Wendy Stock, Nicola Gökbuget, Susan O'Brien, Elias Jabbour, Ryan D. Cassaday, Melanie R. Loyd, Scott Olsen, Geoffrey Neale, Xueli Liu, Erik Vandendries, Charles G. Mullighan, Anjali Advani. Exploration of association of leukemic molecular profile with efficacy in patients (pts) with relapsed/refractory acute lymphoblastic leukemia (ALL) treated with inotuzumab ozogamicin (InO) in the phase 3 INO-VATE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT027.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

The EHA Research Roadmap: Malignant Lymphoid Diseases.