Lineage Acute Lymphoblastic Leukemia Research Articles

B Lineage Acute Lymphoblastic Leukemia with concurrent IGH and EPOR rearrangements – An Entity with Dismal Outcome

B Lineage Acute Lymphoblastic Leukemia with concurrent IGH and EPOR rearrangements – An Entity with Dismal Outcome

Event Free Survival and Overall Survival in Adolescents with Acute Lymphoblastic Leukemia: A Retrospective Review

Aim The therapeutic progress achieved with pediatric regimens in childhood acute lymphoblastic leukemia (ALL) has contributed to the increase of their use in adolescents and young adults (AYA) worldwide, in an attempt to achieve a better event free survival and overall survival in AYA’s with ALL. The outcome of AYA’s with ALL has not been published in Lebanon, neither has the therapy related complications, morbidity and mortality rate. Hence, we decided to conduct a retrospective chart review to include all adolescents aged 12-18 years old, diagnosed with ALL, treated at Children’s Cancer Center of Lebanon, based on the “CCCL/ALL I Study for newly diagnosed patients with Acute Lymphoblastic Leukemia”, enrolled since October 2011 until March 2019. Materials and Methods A retrospective chart review was carried on all adolescents aged 12-18 years old, diagnosed with ALL, treated at Children’s Cancer Center of Lebanon, based on the “CCCL/ALL I Study for newly diagnosed patients with Acute Lymphoblastic Leukemia”, enrolled since October 2011 until March 2019. Data collected included patient’s characteristics (age, gender), leukemia characteristics, MRD at different times, complications during treatment and up to 3 years after ending treatment, in addition to mortality, if present and its causes. Results A total of 38 charts were reviewed, of which 66% of those were males, 79% had B lineage ALL, 13% had T-lineage ALL and 8% had biphenotypic leukemia; none of the patients had CNS disease upon presentation, 3% had testicular involvement upfront. 68% of the patients were stratified as intermediate risk patients, 19% as low risk and 13% as high-risk patients according to the St. Jude consortium classification. With a median follow-up of 5 years, the overall survival was 86%, the relapse rate was 10%. The incidence of pancreatitis and thromboembolic complications was 36% and 30%, respectively. The rate of AVN reached 42% in our patients. Conclusion Adolescents diagnosed with acute lymphoblastic leukemia, in Lebanon, between October 2011 and March 2019, treated as per the CCCL/ALL I Study for newly diagnosed patients with Acute Lymphoblastic Leukemia, based on the SJCRH total XV protocol, showed a similar survival rate in comparison to similarly aged patients treated with other pediatric ALL protocols, worldwide. The complication rate was similar as well, though our patients tended to have a higher incidence of AVN, up to 42%, almost doubling the incidence of AVN in published outcomes of similarly aged patients.

Diagnostic significance of cerebrospinal fluid flow cytometry in Chinese children with B lineage acute lymphoblastic leukemia

BackgroundCentral nervous system leukemia (CNSL) is one of the major causes of the poor prognosis of childhood leukemia. We aimed to compare the sensitivity of cytomorphology (CM) and flow cytometry (FCM) in diagnosing CNSL, emphasizing the importance of FCM in the diagnosis process.MethodsOne-hundred-sixty-five children with newly diagnosed B-cell Acute Lymphoblastic Leukemia (B-cell ALL) were included in this study. Cerebrospinal fluid (CSF) samples were taken for routine CSF analysis, CM analysis, and FCM examination. Computed tomography scans and/or magnetic resonance imaging were performed at diagnosis. Patients with CNS2, CNS3, and traumatic lumbar puncture (TLP) at diagnosis received two additional courses of triple intrathecal injections during induction treatment. We compared the sensitivity of FCM and CM in the diagnosis of children with CNSL.ResultsOne hundred and twenty-eight (77.58%) CSF samples were negative by either CM or FCM (CM−/FCM−), four (2.42%) were positive by both CM and FCM (CM+/FCM+), and thirty-three (20%) displayed a single positive finding by FCM (CM−/FCM+) (p = 0.044). By adding two intrathecal injections in the induction treatment, ten children with TLP+ had no CNS relapse, like those with TLP−. However, compared to CNS1 and TLP, the event-free survival (EFS) did not significantly improve in patients with CNS2 and CNS3. Moreover, CNSL status was associated with worse 3-year EFS (p < 0.05).ConclusionsWe have validated that FCM is more accurate in stratifying the status of the CNS compared to CM analysis. However, to improve the EFS rate of childhood leukemia, it is necessary to combine CM examination, FCM, and cranial imaging for the early diagnosis of CNSL.

Histidine re-sensitizes pediatric acute lymphoblastic leukemia to 6-mercaptopurine through tetrahydrofolate consumption and SIRT5-mediated desuccinylation

Despite progressive improvements in the survival rate of pediatric B-cell lineage acute lymphoblastic leukemia (B-ALL), chemoresistance-induced disease progression and recurrence still occur with poor prognosis, thus highlighting the urgent need to eradicate drug resistance in B-ALL. The 6-mercaptopurine (6-MP) is the backbone of ALL combination chemotherapy, and resistance to it is crucially related to relapse. The present study couples chemoresistance in pediatric B-ALL with histidine metabolism deficiency. Evidence was provided that histidine supplementation significantly shifts the 6-MP dose-response in 6-MP-resistant B-ALL. It is revealed that increased tetrahydrofolate consumption via histidine catabolism partially explains the re-sensitization ability of histidine. More importantly, this work provides fresh insights into that desuccinylation mediated by SIRT5 is an indispensable and synergistic requirement for histidine combination therapy against 6-MP resistance, which is undisclosed previously and demonstrates a rational strategy to ameliorate chemoresistance and protect pediatric patients with B-ALL from disease progression or relapse.

Transcriptome analysis of primary adult B-cell lineage acute lymphoblastic leukemia identifies pathogenic variants and gene fusions, and predicts subtypes for in depth molecular diagnosis.

B-cell acute lymphoblastic leukemia (B-ALL) is classified into subgroups based on known driver oncogenes and molecular lesions, including translocations and recurrent mutations. However, the current diagnostic tests do not identify subtypes or oncogenic lesions for all B-ALL samples, creating a heterogeneous B-ALL group of unknown subtypes. We sorted primary adult B-ALL cells and performed transcriptome analysis by bulk RNA sequencing (RNA-seq). Transcriptomic analysis of an adult B-ALL cohort allowed the classification of four patient samples with subtypes that were not previously revealed by standard gene panels. The leukemia of two patients were of the DUX4 subtype and two were CRLF2+ Ph-like B-ALL. Furthermore, single nucleotide variant analysis detected the oncogenic NRAS-G12D, KRAS-G12D, and KRAS-G13D mutations in three of the patient samples, presenting targetable mutations. Additional oncogenic variants and gene fusions were uncovered, as well as multiple variants in the PDE4DIP gene across five of the patient samples. We demonstrate that RNA-seq is an effective tool for precision medicine in B-ALL by providing comprehensive molecular profiling of leukemia cells, identifying subtype and oncogenic lesions, and stratifying patients for appropriate therapy.

Acute Kidney Injury in Methotrexate treated Leukemia

Background: The study was performed to know the incidence of acute kidney injury within 48 hours following High dose Methotrexate administration in children with high-risk B cell Acute Lymphoblastic Leukemia along with its risk factors and toxicities. Most studies have been conducted in adults and have included non-Hodgkin lymphoma along with leukemia. There is a paucity of data regarding the burden of Acute kidney injury with Methotrexate use in the pediatric age group with B cell Acute Lymphoblastic Leukemia. Methods: It was a prospective observational study conducted at the pediatric ward of All India Institute of Medical Sciences, New Delhi between 2019 September to 2021 June. Children between 1 to 18 years with high-risk B lineage Acute Lymphoblastic Leukemia planned for high-dose Methotrexate at 3gm/m² were enrolled. The measurement of serum creatinine was done at baseline, 24 and 42 hours while serum methotrexate at 24 and 42 hours, adverse reactions were assessed till day 14 of methotrexate administration. Results: The incidence of Acute Kidney Injury and severe Acute Kidney Injury (stages 2 and 3) at 42 hours of High dose Methotrexate infusion was 24.3% and 12.8% respectively. Low serum albumin was associated with an increased risk of Acute Kidney Injury (p=0.004). Transaminitis and thrombocytopenia were common toxicities. Conclusions: Acute Kidney Injury is common following High dose Methotrexate administration in B cell Acute Leukemic Leukemia. Low serum albumin level increases renal toxicity following methotrexate infusion. The measures to prevent renal toxicity should be instituted promptly in high-risk groups.

Novel chimeric antigen receptor targets and constructs for acute lymphoblastic leukemia: Moving beyond CD19.

Acute lymphoblastic leukemia (ALL) is the second most common acute leukemia in adults with a poor prognosis with relapsed or refractory (R/R) B-cell lineage ALL (B-ALL). Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has shown excellent response rates in RR B-ALL, but most patients relapse due to poor persistence of CAR T-cell therapy or other tumor-associated escape mechanisms. In addition, anti-CD19 CAR T-cell therapy causes several serious side effects such as cytokine release syndrome and neurotoxicity. In this review, we will discuss novel CAR targets, CAR constructs, and various strategies to boost CARs for the treatment of RR B-ALL. In addition, we discuss a few novel strategies developed to reduce the side effects of CAR.

Cutaneous Mucormycosis Due to Rhizopus oryzae in Immunocompromised Pediatric Patient

Abstract Introduction Mucormycosis is an infection predominantly in immunocompromised patients by fungi of the Mucorales order, the most frequent being Rhizopus spp., Mucor spp. and Lichteimia spp. Characterized by vascular invasion, necrosis in the lung, CNS, paranasal sinuses, gastrointestinal system, and skin. Case Presentation 2-year-old female patient, native and resident of Valle de Chalco, State of Mexico. Recent diagnosis of B lineage acute lymphoblastic leukemia, in remission induction phase. Medical/surgical history: December 10 2021: Bone marrow aspirate -January 10 2022: Remission induction chemotherapy with vincristine and daunorubicin -January 17 2022: Central venous catheter placement -Family history: Parents are reported healthy. Positive drug addiction for smoking. They have two doses of vaccine for SARS COV2 -Epidemiologic history: Protective isolation was indicated in this patient -Allergies, rash, transfusion and traumatic: Denied -Adequate nutrition, adequate hygiene, complete vaccination schedule. Neurodevelopment according to age -Hospitalized on January 17 for fever and neutropenia, treatment with fourth-generation cephalosporin as initial management. -On January 20, 2022, she presented an erythematousviolaceous papular lesion, raised in the center, without central necrosis, without elevation, and painful on palpation on the right forearm -Treatment with fourth generation cephalosporin and glycopeptide was started, a biopsy culture of the lesion was taken and Central and peripheral blood cultures -On physical examination, an erythematous-violaceous maculopapular lesion was observed on the right forearm, without necrosis, without exudate and painful on palpation. Vital Signs in normal ranges. Relevant labs Leukopenia was found with profound neutropenia and thrombocytopenia. Positive acute phase reactants, with positive procalcitonin and C-reactive protein. Final Diagnosis For the study of the tissue, debridement of the wound, samples of the spindle center and external edges for biopsy and Lactophenol cotton blue staining were performed. Final diagnosis is Cutaneous Mucormycosis. The culture reports Rhizopus oryzae. For treatment, start Liposomal Amphotericin B 5 milligrams per kilogram per dose every 24 hours. It was increased to 10 milligrams per kilogram per dose for progression of the lesion. Teaching Points Mucormycosis is produced by opportunistic fungi of the order Mucorales. It presents an acute and fatal course that requires early diagnosis and treatment to avoid complications and death (1) A timely diagnosis is very important, since with surgical and antifungal treatment the survival rate increases to 70% (2) Isavuconazole showed activity against mucormycosis with efficacy similar to Amphotericin B. Isavuconazole can be used for the treatment of mucormycosis and is well tolerated (3,4).

Epigenomic mapping reveals distinct B cell acute lymphoblastic leukemia chromatin architectures and regulators

B cell lineage acute lymphoblastic leukemia (B-ALL) is composed of diverse molecular subtypes, and while transcriptional and DNA methylation profiling has been extensively examined, the chromatin landscape is not well characterized for many subtypes. We therefore mapped chromatin accessibility using ATAC-seq in primary B-ALL cells from 156 patients spanning ten molecular subtypes and present this dataset as a resource. Differential chromatin accessibility and transcription factor (TF) footprint profiling were employed and identified B-ALL cell of origin, TF-target gene interactions enriched in B-ALL, and key TFs associated with accessible chromatin sites preferentially active in B-ALL. We further identified over 20% of accessible chromatin sites exhibiting strong subtype enrichment and candidate TFs that maintain subtype-specific chromatin architectures. Over 9,000 genetic variants were uncovered, contributing to variability in chromatin accessibility among patient samples. Our data suggest that distinct chromatin architectures are driven by diverse TFs and inherited genetic variants that promote unique gene-regulatory networks.

Exploratory study on the efficacy of bortezomib combining mitoxantrone or CD22-CAR T therapy targeting CD19-negative relapse after CD19-CAR T cell therapy with a simpler cell-line-based model.

Target-negative relapse after CD19 chimeric antigen receptor engineered (CAR) T cell therapy for patients with B lineage acute lymphoblastic leukemia (B-ALL) presents limited treatment options with dismal outcomes. Although CD22-CAR T cells mediate similarly potent antineoplastic effects in patients with CD19dim or even CD19-negative relapse following CD19-directed immunotherapy, a high rate of relapse associated with diminished CD22 cell surface expression has also been observed. Therefore, it is unclear whether any other therapeutic options are available. Mitoxantrone has shown significant antineoplastic activity in patients with relapsed or refractory leukemia over the past decades, and in some cases, the addition of bortezomib to conventional chemotherapeutic agents has demonstrated improved response rates. However, whether this mitoxantrone and bortezomib combination therapy is effective for those patients who have relapsed B-ALL after receiving CD19-CAR T cell therapy remains to be elucidated. In this study, we established a cellular model system using a CD19-positive B-ALL cell line Nalm-6 to investigate the treatment options for CD19-negative relapsed B-ALL after CD19-CAR T cell therapy. In addition to CD22-CAR T therapy, we observed that the combination of bortezomib and mitoxantrone exhibited effective anti-leukemia activity in the CD19-negative Nalm-6 cell line by downregulating p-AKT and p-mTOR. These results suggest that this combination therapy is a possible option for target-negative refractory leukemia cells after CAR-T cell treatment.

Management of B-cell lineage acute lymphoblastic leukemia: expert opinion from an Indian panel via Delphi consensus method.

Currently, there are no guidelines for the management of B-cell lineage acute lymphoblastic leukemia (B-ALL) from an Indian perspective. The diagnostic workup, monitoring, and treatment of B-ALL vary among different physicians and institutes. To develop evidence-based practical consensus recommendations for the management of B-ALL in Indian settings. Modified Delphi consensus methodology was considered to arrive at a consensus. An expert scientific committee of 15 experts from India constituted the panel. Clinically relevant questions belonging to three major domains were drafted for presentation and discussion: (i) diagnosis and risk assignment; (ii) frontline treatment; and (iii) choice of therapy (optimal vs. real-world practice) in relapsed/refractory (R/R) settings. The questionnaire was shared with the panel members through an online survey platform. The level of consensus was categorized into high (≥ 80%), moderate (60%-79%), and no consensus (< 60%). The process involved 2 rounds of discussion and 3 rounds of Delphi survey. The questions that received near or no consensus were discussed during virtual meetings (Delphi rounds 1 and 2). The final draft of the consensus was emailed to the panel for final review. Experts recommended morphologic assessment of peripheral blood or bone marrow, flow cytometric immunophenotyping, and conventional cytogenetic analysis in the initial diagnostic workup. Berlin-Frankfurt-Münster (BFM)-based protocol is the preferred frontline therapy in pediatric and adolescent and young adult patients with B-ALL. BFM/German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia-based regimen is suggested in adult patients with B-ALL. Immunotherapy (blinatumomab or inotuzumab ozogamicin) followed by allogeneic hematopoietic cell transplantation (allo-HCT) is the optimal choice of therapy that would yield the best outcomes if offered in the first salvage in patients with R/R B-ALL. In patients with financial constraints or prior allo-HCT (real-world practice) at first relapse, standard-intensive chemotherapy followed by allo-HCT may be considered. For subsequent relapses, chimeric antigen receptor T-cell therapy or palliative care was suggested as the optimal choice of therapy. This expert consensus will offer guidance to oncologists/clinicians on the management of B-ALL in Indian settings.

Neurofibromatosis Symptom-Lacking B-Cell Lineage Acute Lymphoblastic Leukemia with Only an NF1 Gene Pathogenic Variant

Next-generation sequencing technology has improved molecular genetic analysis, and many molecular genetic studies have been utilized for diagnostic classification, risk stratification, and prognosis prediction of acute lymphoblastic leukemia (ALL). Inactivation of neurofibromin or Nf1, a protein derived from the NF1 gene, causes Ras pathway regulation failure, which is related to leukemogenesis. Pathogenic variants of the NF1 gene in B-cell lineage ALL are uncommon, and in this study, we reported a pathogenic variant that is not registered in any public database. The patient diagnosed with B-cell lineage ALL had no clinical symptoms of neurofibromatosis. Studies on the biology, diagnosis, and treatment of this uncommon disease, as well as other related hematologic neoplasms, such as acute myeloid leukemia and juvenile myelomonocytic leukemia, were reviewed. Biological studies included epidemiological differences among age intervals and pathways for leukemia, such as the Ras pathway. Diagnostic studies included cytogenetic, FISH, and molecular tests for leukemia-related genes and ALL classification, such as Ph-like ALL or BCR-ABL1-like ALL. Treatment studies included pathway inhibitors and chimeric antigen cell receptor T-cells. Resistance mechanisms related to leukemia drugs were also investigated. We believe that these literature reviews will enhance medical care for the uncommon diagnosis of B-cell lineage ALL.

Relapses Children's Acute Lymphoblastic Leukemia, Single Center Experience.

125 pediatric patients with ALL diagnosed in our department between 2000-2018; age 1-18 years old (median 6.4); female 53.6% vs. male 46.4%. 19 pts (15.2%) were diagnosed with a relapse. Three pts (15.8%) had been diagnosed with very early relapses (2/3 T-ALL), 12 pts (63.1%) as an early relapse, and 4 pts (21.1%) as a late relapse. Bone marrow was the most frequent relapses localization. The five-year survival has been achieved by six patients (31.6%). A significant difference was found in regard to the five-year overall survival and relapse type (p &lt; 0.05). The group with very early relapses (3/3; 100%) has not reached the five-year survival. 1. The main prognostic factor in children's ALL relapses is still the time of the onset of the relapse. 2. The T lineage acute lymphoblastic leukemia is a worse prognostic factor. 3. The analysis of the above relapse risk factors alongside cytogenethic markers and flow cytometry or polymerase chain reaction minimal residual disease is very important for first-line chemotherapy improvement and a more personalized choice of therapy for ALL patients.

Efficacy and Safety of Nelarabine in Different Age Groups with T-Cell Hematological Malignancies: A Systematic Review of Clinical Trials

Background: The survival rates of patients with T-cell malignancies especially acute lymphoblastic leukemia (ALL) have significantly improved in the last decade. The survival rates of T- cell lineage ALL are relatively inferior to survival rates of B-cell lineage ALL. Nelarabine is a prodrug for araguanosine that metabolizes into arabinosylguanine nucleotide triphosphate and acts as a DNA terminating nucleoside. In this review, we assessed the efficacy and safety of nelarabine in T-cell hematological malignancies in different age groups of patients. Methods: A literature search was performed on PubMed, Web of Science, Embase (OVID), and clinicaltrials.gov with Mesh terms for, "Nelarabine" AND "Leukemia OR Lymphoma" from the inception of data till June 10, 2022. Out of 1,051 articles, we included 4 randomized clinical trials (RCTs, N=980) and 2 non-randomized clinical trials (nRCTs, N=246) based on the inclusion criteria. We excluded all case reports, review articles, retrospective studies, pre-clinical studies, and meta-analyses. PRISMA guidelines were followed in this systematic review. Results: 2 clinical trials (N=812) included children and young adult patients, 3 clinical studies (N=321) included adult patients and 1 clinical study (N=93) included both children and adults. 1,102 patients had T-cell ALL, 35 had T-cell non-Hodgkin lymphoma, 11 had T-cell pleomorphic lymphoma, and 56 had other hematological malignancies. 945 patients had a newly diagnosed (ND) malignancy while 281 had relapsed/refractory (RR) malignancy. Nelarabine was given to 740 patients while 486 were treated with standard therapy. In an RCT on pediatric patients with ND T-cell ALL (N=659), 5-year disease-free survival (DFS), overall survival (OS), and relapse rate (RR) were 88.2%, 90.3%, and 1.3%, respectively, in patients treated with nelarabine + standard therapy, vs. 82.1%, 87.9%, and 6.9%, respectively, in patients treated with standard therapy. In a trial on RR T-cell ALL/non-Hodgkin (NH) pediatric patients (N=153), the overall response rate (ORR) was 25.7% and complete response (CR) was 29%. In two RCTs (N=286) on adult patients with ND T-cell ALL, 3-year OS and CR were 65% and 87-93%, respectively, in patients treated with nelarabine + standard therapy vs. 3-year OS and CR of 61.7-64% and 88-90.7%, respectively in patients treated standard therapy only. In phase I clinical trial (N=35) on adult patients with RR indolent leukemias, CR and ORR were 4% and 15%, respectively in patients treated with nelarabine and CR and ORR were 11% and 66% in patients treated with nelarabine + fludarabine. In an RCT by Kurtzberg et al. (N=93) on pediatric and adult patients with RR malignancies, CR was 11% and ORR was 31%. Neurotoxicity, infection, hypokalemia, ALT/AST elevation, malaise, and confusion were the common ≥ Grade 3 adverse events reported in patients with T-cell malignancies treated with nelarabine. Table 1, 2. Conclusion: Nelarabine was well tolerated by most of the patients with T-cell malignancies. Nelarabine addition to standard therapy was significantly more effective than placebo in ND T-ALL pediatric patients. However, the addition of nelarabine to standard therapy in adult patients with ND T-ALL did not improve survival or response rates. Nelarabine was also effective in pediatric and adult patients with RR T-ALL, T-NHL, and T-cell indolent leukemias in early phase clinical trials. More randomized double-blind multicenter clinical trials are needed to confirm these results. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of Minimal Residual Disease in B-Acute Lymphoblastic Leukaemia Using Uniform Manifold Approximation Projection

Background and objective: Acute Lymphoblastic Leukaemia (ALL) is the most common childhood cancer, with B lineage ALL accounting for approximately 85% of cases. Minimal residual disease (MRD) levels during induction therapy are important prognostic biomarkers that allow patient stratification dependent on their risk of relapse. MRD is quantified by molecular analyses of antigen receptor gene rearrangements or by flow cytometric evaluation of aberrant immunophenotypes. The latter method is increasingly being used worldwide, due to its reduced cost, its provision of predictive biomarkers for antigen therapies and the inclusion of both MRD methodologies in contemporary clinical trials. However, accurate detection of MRD by flow cytometry is complex, time-consuming and relies heavily on user training and expertise. Machine learning has been evolving in the field, with the aim of superseding manual analysis of Flow MRD data. This study aimed to test the ability of the unsupervised machine learning dimensional reduction algorithm, Uniform Manifold Approximation Projection, (UMAP) to visualise and accurately quantify 'MRD islands' from normal cell populations with minimal human intervention. Methods: Flow cytometric data from bone marrow samples from children entered on the UKALL2011 clinical trial were used in the study. For B lineage ALL, the protocol included the identification of a leukaemia associated immunophenotype at diagnosis using 2 antibody combinations and 'on treatment' samples were then ran with the most appropriate antibody tube. In this study, we used data from Euro tube 1 which included the antibodies, CD20 FITC, CD38 PE, CD34 PerCP, CD10 PE-Cy7, CD19 APC, CD45 APC-Cy7 and the nuclear dye, Syto 41, tagged with Pacific Blue. FCS files were uploaded to Cytobank and pre-gated for CD19 positive cells prior to running the UMAP algorithm. Each data set was ran alongside data from 10 normal marrows stained with Euro tube 1. Results: Initially, a selection of samples with varying MRD levels (n=6) were ran using UMAP with a range of minimum distance and number of neighbours parameters. A minimum distance of 0.1 and number of neighbours of 13 was the optimal UMAP settings to produce a very clear separation of MRD islands from healthy cells populations across all samples. (Figure 1). These setting were then used for 341 B-ALL MRD samples at different time points day 8 (n=116), day 15 (n=32), days 29 (n=118), weeks 6-20 (n= 75). MRD levels from UMAP were compared to those generated by the gold standard sequential gating. Excellent concordance was seen, as demonstrated by an R2 value of 0.99 (linear correlation). For 'on treatment samples', UMAP showed excellent accuracy with sensitivity and specificity values of 93% and 100%, respectively. Positive predictive values were 100% while negative predictive values were 94% due to UMAP analysis failing to identify an MRD island in 11 'on treatment' samples, 9 at day 8 and 2 at day 28. The average UMAP run time was less than 2 minutes. Conclusion: Detection of MRD using the UMAP algorithm showed an excellent correlation with the gold standard sequential gating technique. False negatives were due to overlap of antigen expression with normal cells and/or low ALL cell numbers. Current Flow MRD methodologies use 8 or more antibodies and higher target cell numbers, which may reduce the false negative UMAP call rate. In summary, UMAP has promising ability to simplify the downstream data analyses of Flow MRD, overcoming the extensive training needed, human subjectivity and will be time and thus cost saving. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chromatin Accessibility Landscapes of B-Cell Acute Lymphoblastic Leukemia Identify Extensive Epigenomic Reprogramming and Heterogeneity Among Subtypes

Acute lymphoblastic leukemia (ALL) is the most common malignancy in pediatric patients and represents about a quarter of all pediatric malignancies. Past work has characterized B-cell lineage ALL (B-ALL) into molecular subtypes spanning a range of aberrant chromosomal rearrangements and oncogene chimeric fusions driving malignancy. While transcriptional and DNA methylome profiling of these subtypes has been extensively examined, the accompanying chromatin landscape and corresponding gene regulatory repertoire are not well characterized for many B-ALL subtypes. To better profile the B-ALL epigenome and gene regulatory networks we examined chromatin accessibility of 11 distinct molecular subtypes (BCR-ABL1, DUX4/ERG, ETV6-RUNX1, Hyperdiploid, Low hypodiploid, KMT2A-rearranged, Ph-like, PAX5-altered, TCF3-PBX1, ZNF384-rearranged and B-other) using 154 primary patient samples from across the United States with assay for transposase-accessible chromatin and high-throughput sequencing (ATAC-seq). We mapped extensive chromatin reprogramming when comparing B-ALL and normal B-cells that drive oncogenic signaling pathways and inhibit normal B-cell functions. Strikingly, we also uncovered chromatin accessibility sites in B-ALL (42,457 sites) with distinct subtype-specific enrichment. A classification model utilizing subtype distinct loci was able to accurately predict B-ALL subtypes with 87% cross-validation accuracy. Transcription factor (TF) footprint profiling and transcriptomic information further identified candidate subtype-enriched TFs, such as DUX4, ZNF384, HOXA9, MEIS1, GATA3 and RARA, along with subtype distinct gene regulatory networks. In connecting these findings with genetic variation, we identified 9080 inherited DNA sequence variants that act as ATAC-seq chromatin accessibility quantitative trait loci (ATAC-QTLs) and contribute to variability in chromatin accessibility among patient samples. Nearly one-third of ATAC-QTLs map to TF footprints and commonly altered DNA consensus motifs impacting chromatin accessibility include ETS family TFs (PU.1, SPI-B, ELF3, EHF), ZNF family (ZNF263, ZNF460) and CTCF. Collectively, using the largest B-ALL chromatin accessibility dataset to date, our investigation offers a unique window into the gene regulatory landscape that highlights the complexity and heterogeneity of chromatin accessibility among B-ALL molecular subtypes.

Clinical and Cytogenetic Characteristics of Children With Leukemia 20-Year Retrospective Study.

Acute leukemias are the most common malignant diseases in childhood. The aims of this retrospective cohort study were to investigate the frequency of cytogenetic abnormalities in acute pediatric leukemia; the correlation between cytogenetic abnormalities and 5-year survival; and the correlation between cytogenetic abnormalities and clinical and laboratory features. We included 105 patients; acute lymphoblastic leukemia (ALL) had 80.9% patients, B-cell lineage ALL (B-ALL) 84.7% of them, and T-cell lineage (T-ALL) 15.3%. The overall 5-year survival for B-ALL was 85.9% and for T-ALL was 84.6%. The most common cytogenetic abnormalities in patients with B-ALL were t(12;21)(p13.2;q22.1); ETV6-RUNX1 with 22.2% and hyperdiploidy with 19.4%. Our survival analysis showed that t(12;21)(p13.2;q22.1); ETV6-RUNX1 and t(1;19)(q23;p13.3); TCF3-PBX1 had the best 5-year survival with 100% of patients surviving, whereas t(v;11q23.3); KMT2A rearranged had the worst 5-year survival of just 33.3% of patients surviving after 5 years. We found no difference in 5-year survival in B-ALL when comparing clinical features. Acute myelogenous leukemia had 20 patients with 70.6% 5-year survival. The most common cytogenetic abnormality in acute myelogenous leukemia was t(8;21)(q21;q22.1); RUNX1-RUNX1T1 (20%). In conclusion, this study showed the correlation of different cytogenetic abnormalities with 5-year survival in B-ALL patients. Such correlation was not found when comparing clinical features and 5-year survival of patients with B-ALL. This emphasized the significance of cytogenetic analysis in pediatric leukemia.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

Role of chimeric antigen receptor T-cell therapy: bridge to transplantation or stand-alone therapy in pediatric acute lymphoblastic leukemia.

To discuss the curative potential for chimeric antigen receptor T-cell (CAR-T) therapy, with or without consolidative hematopoietic stem cell transplantation (HCT) in the treatment of children and young adults with B lineage acute lymphoblastic leukemia (B-ALL). CAR-T targeting CD19 can induce durable remissions and prolong life in patients with relapsed/refractory B-ALL. Whether HCT is needed to consolidate remission and cure relapse/refractory B-ALL following a CD19 CAR-T induced remission remains controversial. Preliminary evidence suggests that consolidative HCT following CAR-T in HCT-naïve children improves leukemia-free survival. However, avoiding HCT-related late effects is a desirable goal, so identification of patients at high risk of relapse is needed to appropriately direct those patients to HCT when necessary, while avoiding HCT in others. High disease burden prior to CAR-T infusion, loss of B-cell aplasia and detection of measurable residual disease by flow cytometry or next-generation sequencing following CAR-T therapy associate with a higher relapse risk and may identify patients requiring consolidative HCT for relapse prevention. There is a pressing need to determine when CD19 CAR-T alone is likely to be curative and when a consolidative HCT will be required. We discuss the current state of knowledge and future directions.

Stat5 Feedback Control Switches MYC- and BCL6-Dependent Cell Fates in Normal and Transformed B Cells

Background: During early B cell development, IL7-dependent large cycling pre-B cells undergo dramatic phenotypic changes and differentiate into small resting pre-B cells in the absence of IL7. Inhibition of oncogenic tyrosine kinases in pre-B acute lymphoblastic leukemia (ALL) results in very similar changes. Both IL7 cytokine signaling in pre-B cells and oncogenic tyrosine kinase activity in pre-B ALL induces strong activation of Stat5. STAT5 induces its own negative feedback through transcriptional activation of CISH and SOCS proteins. Interestingly, SOCS and CISH are highly expressed in pre-B ALL cells and represent the focus of this study.Results: mRNA levels of SOCS2 and CISH were 10-fold and 3-fold upregulated in ALL compared to normal pre-B cells respectively. Importantly, we found that high expression levels of SOCS/CISH are predictive of poor outcome in one clinical trial for ALL patients. These results indicate that inhibitory signaling from SOCS2 and CISH may contribute to the disease progression of ALL. To study the function of Socs2, Socs3 and Cish in genetic experiments, we transformed pre-B cells from mutant mice. Deletion of Socs2, Socs3 or Cish strongly increased phosphorylation of Stat5, increased ROS and accumulation of Arf, p53 and p21, ultimately resulting in leukemia cell death. Studying BCR-ABL1 transformed Cishfl/fl ALL cells in vivo, we found that genetic deletion of Cish resulted in rapid leukemia regression and prolonged survival of recipient NOD-SCID mice (13 days vs 30 days; n=7; p<0.001). To elucidate the underlying mechanisms, we transduced a constitutively active form of STAT5 (Stat5CA) or a non-phosphorylatable form of STAT5 (Stat5Y694F) into B cell lineage ALL cells. While Stat5CA increased cell size by nearly 2 fold, Stat5Y694F caused rapid cell shrinkage. RNA-seq and Western blot results showed that Stat5CA increased MYC and glycolytic gene expression but decreased BCL6 and fatty acid oxidation (FAO)-related genes. Conversely, Stat5Y694F upregulated BCL6 expression and a FAO-related gene expression program, but decreased MYC and glycolytic gene expression. Metabolomics analyses confirmed that Stat5CA increased glucose consumption, ATP production and protein synthesis. While Stat5Y694F decreased glucose consumption, ATP production, and increased fatty acid oxidation through BCL6 and AMPK-activation. ChIP-seq revealed both forms of STAT5 can directly bind to MYC and BCL6 promoters as well as glycolytic and FAO targets, albeit with antagonizing effects. Inhibition of mTOR signaling by Rapamycin or protein synthesis inhibitors or knocking down MYC rescued Stat5CA-induced toxicity in the context of dramatically enlarged cell size. On the other hand, overexpression of MYC rescued Stat5Y694F-transduced cells by increasing glucose consumption and ATP production in cells that would otherwise dramatically shrink.Conclusion: Our results suggest that STAT5 feedback control is required to keep a balance between p-STAT5 (CA) and non-p-STAT5 (Y694F), which promote divergent, MYC (CA) and BCL6 (Y694F)-dependent transcriptional programs. A single mutation affecting the phosphorylation status of STAT5-Y694 has far reaching impact on cell fate, differentiation status, cell size and metabolic behavior (glycolysis vs FAO), which need to be balanced by STAT5-feedback control. DisclosuresMuschen:AbbVie: Research Funding; Pfizer: Research Funding.