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Acute lymphoblastic leukemia expressing the gamma delta T-cell receptor (γδ T-ALL) is a poorly understood disease. We studied 200 children with γδ T-ALL from 13 clinical study groups to understand the clinical and genetic features of this disease. We found age and genetic drivers were significantly associated with outcome. γδ T-ALL diagnosed in children under 3 years of age was extremely high-risk and enriched for genetic alterations that result in both LMO2 activation and STAG2 inactivation. Mechanistically, using patient samples and isogenic cell lines, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping, resulting in deregulation of gene expression associated with T-cell differentiation. High-throughput drug screening identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which can be targeted by poly(ADP-ribose) polymerase inhibition. These data provide a diagnostic framework for classification and risk stratification of pediatric γδ T-ALL. Significance: Patients with acute lymphoblastic leukemia expressing the gamma delta T-cell receptor under 3 years old or measurable residual disease ≥1% at end of induction showed dismal outcomes and should be classified as having high-risk disease. The STAG2/LMO2 subtype was enriched in this very young age group. STAG2 inactivation may perturb chromatin conformation and cell differentiation and confer vulnerability to poly(ADP-ribose) polymerase inhibition., (©2024 American Association for Cancer Research.)

Several studies reported that patients with acute myeloid leukemia (AML) who remain in long-term remission after allogeneic or autologous transplant have a shorter life expectancy, compared to the general population. However, little is known about the life expectancy of adult long-term survivors of AML who were treated with chemotherapy alone without a transplant and there have been no comparisons with survival among the general population. The current study indicates that the life expectancy of AML patients who achieved and maintained CR for at least 3 years is shorter than expected for age in the US population. This was observed also in patients who did not undergo a transplant including those who have not relapsed during the entire long follow-up period. Thus, late relapse does not explain why patients without transplants have a shortened life expectancy. Taken together, these data strongly suggest that prior chemotherapy for the underlying AML is at least a major contributing factor for the known shortened life expectancy post-transplant., (© 2024. The Author(s).)

Introduction: Relapsed or refractory (r/r) acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LL) remains a therapeutic challenge. Preclinical data in both B- and T-ALL suggests synergy of venetoclax (VEN) with vincristine (VCR). We designed a phase I/II trial (EA9152) of the combination of L-VCR and VEN for patients with r/r B-or T-cell ALL or LL. Here, we report the safety and efficacy outcomes of the phase I portion of this trial (NCT03504644)., Methods: In a 3+3 dose escalation design, r/r ALL subjects were given single-agent VEN doses reaching 400, 600, or 800 mg for the three respective dose levels. Weekly L-VCR at 2.25 mg/m 2 IV was started on D15 of cycle 1. The primary phase I objective was to determine the maximum tolerated dose (MTD) of the combination., Results: Among the 18 patients in phase I, grade ≥ 3 treatment-related adverse events were reported in 89% of treated patients. Two patients (two of three) at dose level 3 experienced dose-limiting toxicities. Therefore, the MTD of the combination was determined to be dose level 2 (VEN 600 mg). Twenty-two percent of evaluable patients ( N  = 4) achieved a complete response, with two of them showing no evidence of measurable residual disease (MRD)., Conclusion: The combination of VEN and L-VCR was found to be safe for patients with r/r ALL and encouraging preliminary efficacy, including MRD negative responses. With the removal of L-VCR from the US market, the phase 2 portion of this trial is actively enrolling with vincristine sulfate., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Background: Many older adults with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have a relapse despite having a measurable residual disease (MRD)-negative complete remission with combination chemotherapy. The addition of blinatumomab, a bispecific T-cell engager molecule that is approved for the treatment of relapsed, refractory, and MRD-positive BCP-ALL, may have efficacy in patients with MRD-negative remission., Methods: In a phase 3 trial, we randomly assigned patients 30 to 70 years of age with BCR::ABL1 -negative BCP-ALL (with :: indicating fusion) who had MRD-negative remission (defined as <0.01% leukemic cells in bone marrow as assessed on flow cytometry) after induction and intensification chemotherapy to receive four cycles of blinatumomab in addition to four cycles of consolidation chemotherapy or to receive four cycles of consolidation chemotherapy alone. The primary end point was overall survival, and relapse-free survival was a secondary end point., Results: The data and safety monitoring committee reviewed the results from the third efficacy interim analysis and recommended that they be reported. Complete remission with or without full count recovery was observed in 395 of 488 enrolled patients (81%). Of the 224 patients with MRD-negative status, 112 were assigned to each group. The characteristics of the patients were balanced between the groups. At a median follow-up of 43 months, an advantage was observed in the blinatumomab group as compared with the chemotherapy-only group with regard to overall survival (at 3 years: 85% vs. 68%; hazard ratio for death, 0.41; 95% confidence interval [CI], 0.23 to 0.73; P = 0.002), and the 3-year relapse-free survival was 80% with blinatumomab and 64% with chemotherapy alone (hazard ratio for relapse or death, 0.53; 95% CI, 0.32 to 0.87). A higher incidence of neuropsychiatric events was reported in the blinatumomab group than in the chemotherapy-only group., Conclusions: The addition of blinatumomab to consolidation chemotherapy in adult patients in MRD-negative remission from BCP-ALL significantly improved overall survival. (Funded by the National Institutes of Health and others; E1910 ClinicalTrials.gov number, NCT02003222.)., (Copyright © 2024 Massachusetts Medical Society.)

Sequencing of bulk tumor populations has improved genetic classification and risk assessment of B-ALL, but does not directly examine intratumor heterogeneity or infer leukemia cellular origins. We profiled 89 B-ALL samples by single-cell RNA-seq (scRNA-seq) and compared them to a reference map of normal human B-cell development established using both functional and molecular assays. Intra-sample heterogeneity was driven by cell cycle, metabolism, differentiation, and inflammation transcriptional programs. By inference of B lineage developmental state composition, nearly all samples possessed a high abundance of pro-B cells, with variation between samples mainly driven by sub-populations. However, ZNF384- r and DUX4- r B-ALL showed composition enrichment of hematopoietic stem cells, BCR::ABL1 and KMT2A -r ALL of Early Lymphoid progenitors, MEF2D -r and TCF3::PBX1 of Pre-B cells. Enrichment of Early Lymphoid progenitors correlated with high-risk clinical features. Understanding variation in transcriptional programs and developmental states of B-ALL by scRNA-seq refines existing clinical and genomic classifications and improves prediction of treatment outcome.

Purpose: Gamma delta T-cell receptor-positive acute lymphoblastic leukemia (γδ T-ALL) is a high-risk but poorly characterized disease., Methods: We studied clinical features of 200 pediatric γδ T-ALL, and compared the prognosis of 93 cases to 1,067 protocol-matched non-γδ T-ALL. Genomic features were defined by transcriptome and genome sequencing. Experimental modeling was used to examine the mechanistic impacts of genomic alterations. Therapeutic vulnerabilities were identified by high throughput drug screening of cell lines and xenografts., Results: γδ T-ALL in children under three was extremely high-risk with 5-year event-free survival (33% v. 70% [age 3-<10] and 73% [age ≥10], P =9.5 x 10 -5 ) and 5-year overall survival (49% v. 78% [age 3-<10] and 81% [age ≥10], P =0.002), differences not observed in non-γδ T-ALL. γδ T-ALL in this age group was enriched for genomic alterations activating LMO2 activation and inactivating STAG2 inactivation ( STAG2/LMO2 ). Mechanistically, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping resulting in deregulation of gene expression associated with T-cell differentiation. Drug screening showed resistance to prednisolone, consistent with clinical slow treatment response, but identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which was efficaciously targeted by Poly(ADP-ribose) polymerase (PARP) inhibition, with synergism with HDAC inhibitors. Ex-vivo drug screening on PDX cells validated the efficacy of PARP inhibitors as well as other potential targets including nelarabine., Conclusion: γδ T-ALL in children under the age of three is extremely high-risk and enriched for STAG2/LMO2 ALL. STAG2 loss perturbs chromatin conformation and differentiation, and STAG2/LMO2 ALL is sensitive to PARP inhibition. These data provide a diagnostic and therapeutic framework for pediatric γδ T-ALL., Support: The authors are supported by the American and Lebanese Syrian Associated Charities of St Jude Children's Research Hospital, NCI grants R35 CA197695, P50 CA021765 (C.G.M.), the Henry Schueler 41&9 Foundation (C.G.M.), and a St. Baldrick's Foundation Robert J. Arceci Innovation Award (C.G.M.), Gabriella Miller Kids First X01HD100702 (D.T.T and C.G.M.) and R03CA256550 (D.T.T. and C.G.M.), F32 5F32CA254140 (L.M.), and a Garwood Postdoctoral Fellowship of the Hematological Malignancies Program of the St Jude Children's Research Hospital Comprehensive Cancer Center (S.K.). This project was supported by the National Cancer Institute of the National Institutes of Health under the following award numbers: U10CA180820, UG1CA189859, U24CA114766, U10CA180899, U10CA180866 and U24CA196173., Disclaimer: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funding agencies were not directly involved in the design of the study, gathering, analysis and interpretation of the data, writing of the manuscript, or decision to submit the manuscript for publication.

The bone marrow microenvironment (BME) drives drug resistance in acute lymphoblastic leukemia (ALL) through leukemic cell interactions with bone marrow (BM) niches, but the underlying mechanisms remain unclear. Here, we show that the interaction between ALL and mesenchymal stem cells (MSCs) through integrin β1 induces an epithelial-mesenchymal transition (EMT)-like program in MSC-adherent ALL cells, resulting in drug resistance and enhanced survival. Moreover, single-cell RNA sequencing analysis of ALL-MSC co-culture identifies a hybrid cluster of MSC-adherent ALL cells expressing both B-ALL and MSC signature genes, orchestrated by a WNT/β-catenin-mediated EMT-like program. Blockade of interaction between β-catenin and CREB binding protein impairs the survival and drug resistance of MSC-adherent ALL cells in vitro and results in a reduction in leukemic burden in vivo. Targeting of this WNT/β-catenin-mediated EMT-like program is a potential therapeutic approach to overcome cell extrinsically acquired drug resistance in ALL., Competing Interests: Declaration of interests I.I. received honoraria from Mission Bio. C.G.M. received research funding from AbbVie and Pfizer, honoraria from Amgen and Illumina, and royalty payments from Cyrus. C.G.M. is on an advisory board for Illumina. N.H. and M.P.V. have patent applications in the field of immunotherapy., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Leukemia cells accumulate DNA damage, but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/1-carbon cycle metabolism contributed to the accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1). To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLθ) via ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLθ and its proteasomal degradation. Overexpression of POLθ in OTK-positive cells resulted in the efficient repair of DPC-containing DNA double-strand breaks by POLθ-mediated end-joining. The transforming activities of OTKs and other leukemia-inducing oncogenes, especially of those causing the inhibition of BRCA1/2-mediated homologous recombination with and without concomitant inhibition of DNA-PK-dependent nonhomologous end-joining, was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLθ polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitors or DPC-inducing drug etoposide enhanced the antileukemia effect of POLθ inhibitor in vitro and in vivo. In conclusion, we demonstrated that POLθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect., (© 2023 by The American Society of Hematology.)

Herein, we present the long-term follow-up of the randomized E1912 trial comparing the long-term efficacy of ibrutinib-rituximab (IR) therapy to fludarabine, cyclophosphamide, and rituximab (FCR) and describe the tolerability of continuous ibrutinib. The E1912 trial enrolled 529 treatment-naïve patients aged ≤70 years with chronic lymphocytic leukemia (CLL). Patients were randomly assigned (2:1 ratio) to receive IR or 6 cycles of FCR. With a median follow-up of 5.8 years, median progression-free survival (PFS) is superior for IR (hazard ratio [HR], 0.37; P < .001). IR improved PFS relative to FCR in patients with both immunoglobulin heavy chain variable region (IGHV) gene mutated CLL (HR: 0.27; P < .001) and IGHV unmutated CLL (HR: 0.27; P < .001). Among the 354 patients randomized to IR, 214 (60.5%) currently remain on ibrutinib. Among the 138 IR-treated patients who discontinued treatment, 37 (10.5% of patients who started IR) discontinued therapy due to disease progression or death, 77 (21.9% of patients who started IR) discontinued therapy for adverse events (AEs)/complications, and 24 (6.8% of patients who started IR) withdrew for other reasons. Progression was uncommon among patients able to remain on ibrutinib. The median time from ibrutinib discontinuation to disease progression or death among those who discontinued treatment for a reason other than progression was 25 months. Sustained improvement in overall survival (OS) was observed for patients in the IR arm (HR, 0.47; P = .018). In conclusion, IR therapy offers superior PFS relative to FCR in patients with IGHV mutated or unmutated CLL, as well as superior OS. Continuous ibrutinib therapy is tolerated beyond 5 years in the majority of CLL patients. This trial was registered at www.clinicaltrials.gov as #NCT02048813., (© 2022 by The American Society of Hematology.)

Transcriptome sequencing has identified multiple subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL) of prognostic significance, but a minority of cases lack a known genetic driver. Here, we used integrated whole-genome (WGS) and -transcriptome sequencing (RNA-seq), enhancer mapping, and chromatin topology analysis to identify previously unrecognized genomic drivers in B-ALL. Newly diagnosed (n = 3221) and relapsed (n = 177) B-ALL cases with tumor RNA-seq were studied. WGS was performed to detect mutations, structural variants, and copy number alterations. Integrated analysis of histone 3 lysine 27 acetylation and chromatin looping was performed using HiChIP. We identified a subset of 17 newly diagnosed and 5 relapsed B-ALL cases with a distinct gene expression profile and 2 universal and unique genomic alterations resulting from aberrant recombination-activating gene activation: a focal deletion downstream of PAN3 at 13q12.2 resulting in CDX2 deregulation by the PAN3 enhancer and a focal deletion of exons 18-21 of UBTF at 17q21.31 resulting in a chimeric fusion, UBTF::ATXN7L3. A subset of cases also had rearrangement and increased expression of the PAX5 gene, which is otherwise uncommon in B-ALL. Patients were more commonly female and young adult with median age 35 (range,12-70 years). The immunophenotype was characterized by CD10 negativity and immunoglobulin M positivity. Among 16 patients with known clinical response, 9 (56.3%) had high-risk features including relapse (n = 4) or minimal residual disease >1% at the end of remission induction (n = 5). CDX2-deregulated, UBTF::ATXN7L3 rearranged (CDX2/UBTF) B-ALL is a high-risk subtype of leukemia in young adults for which novel therapeutic approaches are required., (© 2022 by The American Society of Hematology.)

Early T-cell acute lymphoblastic leukemia (ETP-ALL) is an aggressive hematologic malignancy associated with early relapse and poor prognosis that is genetically, immunophenotypically, and transcriptionally distinct from more mature T-cell acute lymphoblastic leukemia (T-ALL) tumors. Here, we leveraged global metabolomic and transcriptomic profiling of primary ETP- and T-ALL leukemia samples to identify specific metabolic circuitries differentially active in this high-risk leukemia group. ETP-ALLs showed increased biosynthesis of phospholipids and sphingolipids and were specifically sensitive to inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in the mevalonate pathway. Mechanistically, inhibition of cholesterol synthesis inhibited oncogenic AKT1 signaling and suppressed MYC expression via loss of chromatin accessibility at a leukemia stem cell-specific long-range MYC enhancer. In all, these results identify the mevalonate pathway as a druggable novel vulnerability in high-risk ETP-ALL cells and uncover an unanticipated critical role for cholesterol biosynthesis in signal transduction and epigenetic circuitries driving leukemia cell growth and survival., Significance: Overtly distinct cell metabolic pathways operate in ETP- and T-ALL pointing to specific metabolic vulnerabilities. Inhibition of mevalonate biosynthesis selectively blocks oncogenic AKT-MYC signaling in ETP-ALL and suppresses leukemia cell growth. Ultimately, these results will inform the development of novel tailored and more effective treatments for patients with high-risk ETP-ALL. This article is highlighted in the In This Issue feature, p. 587., (©2021 American Association for Cancer Research.)

E1912 was a randomized phase 3 trial comparing indefinite ibrutinib plus 6 cycles of rituximab (IR) to 6 cycles of fludarabine, cyclophosphamide, and rituximab (FCR) in untreated younger patients with CLL. We describe measurable residual disease (MRD) levels in E1912 over time and correlate them with clinical outcome. Undetectable MRD rates (<1 CLL cell per 104 leukocytes) were 29.1%, 30.3%, 23.4%, and 8.6% at 3, 12, 24, and 36 months for FCR, and significantly lower at 7.9%, 4.2%, and 3.7% at 12, 24, and 36 months for IR, respectively. Undetectable MRD at 3, 12, 24, and 36 months was associated with longer progression-free survival (PFS) in the FCR arm, with hazard ratios (MRD detectable/MRD undetectable) of 4.29 (95% confidence interval [CI], 1.89-9.71), 3.91 (95% CI, 1.39-11.03), 14.12 (95% CI, 1.78-111.73), and not estimable (no events among those with undetectable MRD), respectively. In the IR arm, patients with detectable MRD did not have significantly worse PFS compared with those in whom MRD was undetectable; however, PFS was longer in those with MRD levels <10-1 than in those with MRD levels above this threshold. Our observations provide additional support for the use of MRD as a surrogate end point for PFS in patients receiving FCR. In patients on indefinite ibrutinib-based therapy, PFS did not differ significantly by undetectable MRD status, whereas those with MRD <10-1 tended to have longer PFS, although continuation of ibrutinib would very likely be necessary to maintain treatment efficacy., (© 2021 by The American Society of Hematology.)

CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) accounts for more than half of Philadelphia chromosome-like (Ph-like) ALL and is associated with a poor outcome in children and adults. Overexpression of CRLF2 results in activation of Janus kinase (JAK)-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of JAKs show variable and limited efficacy. Here, we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against JAKs. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of a series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers, and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity was tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded JAKs and potently killed CRLF2r cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1 and suppressed proliferation of CRLF2r ALL in vivo, e.g. compound 7 (SJ988497). Although dual JAK/GSPT1-degrading PROTACs were the most potent, the development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading, GSPT1-sparing PROTAC that demonstrated efficacy in the majority of kinase-driven xenografts that were otherwise unresponsive to type I JAK inhibitors, e.g. compound 8 (SJ1008030). Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT-driven ALL and highlight the interplay of JAK and GSPT1 degradation activity in this context., (© 2021 by The American Society of Hematology.)

Central nervous system (CNS) involvement in patients with newly diagnosed acute myeloid leukemia (AML) is rare, and systematic data regarding outcome are scarce. This retrospective study summarized data from 11 consecutive Eastern Cooperative Oncology Group-American College of Radiology Imaging Network (ECOG-ACRIN) clinical trials for patients with newly diagnosed AML. In all, 3240 patients with AML were analyzed, and 36 (1.11%) were found to have CNS involvement at diagnosis. The incidence of CNS disease among the 5 studies with per protocol mandatory lumbar puncture (LP) was similar to the incidence among studies in which LP was performed at the discretion of the investigator (0.86% vs 1.41%; P = .18). There was no significant difference in the rate of complete remission (CR) among patients with CNS involvement and those with other extramedullary disease (EMD) sites or those with no EMD (52.8% vs 59.3%-60%). The median overall survival (OS) for patients who were CNS positive, who had other EMD, or who had no EMD was 11.4, 11.3, and 12.7 months, respectively. There was no difference in OS among patients with CNS involvement, those with other EMD (hazard ratio [HR], 0.96; adjusted P = .84), and those with no EMD (HR, 1.19; adjusted P = .44). In conclusion, the reported incidence of CNS involvement in patients with newly diagnosed AML is low (1.1%), irrespective of whether an LP is mandatory or not. The presence of CNS disease at diagnosis in and of itself does not seem to portend a poor prognosis for achieving an initial CR or for OS., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Lineage-ambiguous leukemias are high-risk malignancies of poorly understood genetic basis. Here, we describe a distinct subgroup of acute leukemia with expression of myeloid, T lymphoid, and stem cell markers driven by aberrant allele-specific deregulation of BCL11B , a master transcription factor responsible for thymic T-lineage commitment and specification. Mechanistically, this deregulation was driven by chromosomal rearrangements that juxtapose BCL11B to superenhancers active in hematopoietic progenitors, or focal amplifications that generate a superenhancer from a noncoding element distal to BCL11B . Chromatin conformation analyses demonstrated long-range interactions of rearranged enhancers with the expressed BCL11B allele and association of BCL11B with activated hematopoietic progenitor cell cis -regulatory elements, suggesting BCL11B is aberrantly co-opted into a gene regulatory network that drives transformation by maintaining a progenitor state. These data support a role for ectopic BCL11B expression in primitive hematopoietic cells mediated by enhancer hijacking as an oncogenic driver of human lineage-ambiguous leukemia. SIGNIFICANCE: Lineage-ambiguous leukemias pose significant diagnostic and therapeutic challenges due to a poorly understood molecular and cellular basis. We identify oncogenic deregulation of BCL11B driven by diverse structural alterations, including de novo superenhancer generation, as the driving feature of a subset of lineage-ambiguous leukemias that transcend current diagnostic boundaries. This article is highlighted in the In This Issue feature, p. 2659 ., (©2021 American Association for Cancer Research.)

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3 ITD , BCR-ABL1, JAK2 V617F , and MPL W515L , or with mutations affecting related signaling pathways such as NRAS G12D and CALR del52 . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo , whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors., (©2021 American Association for Cancer Research.)

There are conflicting reports in the literature suggesting that one gender or the other has a better survival with acute myeloid leukaemia (AML). The present study was done in an attempt to resolve the issue. The effect of gender was examined on 3546 newly diagnosed patients with AML, including 548 patients with acute promyelocytic leukaemia (APL) enrolled in 10 multi-institutional treatment studies from March 1984 to November 2008. Kaplan-Meier estimates were used to estimate event-time distributions for survival and multivariate models were used to examine the gender effect after adjusting for multiple risk factors. P values were based on two-sided tests. Non-APL female patients had a significantly better overall (OS) but not disease-free survival (DFS) than males, irrespective of age, initial white blood cell count, or dose of daunorubicin. No differences were observed for obese or FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)-positive patients. Female patients with APL had a significantly better OS and DFS than male patients with APL, and differences in survival were greater for patients with t(15;17) + other cytogenetic abnormalities compared with those with t(15;17) only. Gender is an independent prognostic variable in patients with AML. Whether these survival differences are due to hormonal, genetic or pharmacokinetic differences between the sexes or differential toxin exposure such as smoking is unknown. However, the former seems less likely as patient age did not influence the survival advantage for female patients., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Patients with acute myeloid leukemia (AML) frequently relapse after chemotherapy, yet the mechanism by which AML reemerges is not fully understood. Herein, we show that primary AML cells enter a senescence-like phenotype following chemotherapy in vitro and in vivo . This is accompanied by induction of senescence/inflammatory and embryonic diapause transcriptional programs, with downregulation of MYC and leukemia stem cell genes. Single-cell RNA sequencing suggested depletion of leukemia stem cells in vitro and in vivo , and enrichment for subpopulations with distinct senescence-like cells. This senescence effect was transient and conferred superior colony-forming and engraftment potential. Entry into this senescence-like phenotype was dependent on ATR, and persistence of AML cells was severely impaired by ATR inhibitors. Altogether, we propose that AML relapse is facilitated by a senescence-like resilience phenotype that occurs regardless of their stem cell status. Upon recovery, these post-senescence AML cells give rise to relapsed AMLs with increased stem cell potential. SIGNIFICANCE: Despite entering complete remission after chemotherapy, relapse occurs in many patients with AML. Thus, there is an urgent need to understand the relapse mechanism in AML and the development of targeted treatments to improve outcome. Here, we identified a senescence-like resilience phenotype through which AML cells can survive and repopulate leukemia. This article is highlighted in the In This Issue feature, p. 1307 ., (©2021 American Association for Cancer Research.)

Histone post-translational modifications (PTMs) create a powerful regulatory mechanism for maintaining chromosomal integrity in cells. Histone acetylation and methylation, the most widely studied histone PTMs, act in concert with chromatin-associated proteins to control access to genetic information during transcription. Alterations in cellular histone PTMs have been linked to disease states and have crucial biomarker and therapeutic potential. Traditional bottom-up mass spectrometry of histones requires large numbers of cells, typically one million or more. However, for some cell subtype-specific studies, it is difficult or impossible to obtain such large numbers of cells and quantification of rare histone PTMs is often unachievable. An established targeted LC-MS/MS method was used to quantify the abundance of histone PTMs from cell lines and primary human specimens. Sample preparation was modified by omitting nuclear isolation and reducing the rounds of histone derivatization to improve detection of histone peptides down to 1,000 cells. In the current study, we developed and validated a quantitative LC-MS/MS approach tailored for a targeted histone assay of 75 histone peptides with as few as 10,000 cells. Furthermore, we were able to detect and quantify 61 histone peptides from just 1,000 primary human stem cells. Detection of 37 histone peptides was possible from 1,000 acute myeloid leukemia patient cells. We anticipate that this revised method can be used in many applications where achieving large cell numbers is challenging, including rare human cell populations., Competing Interests: The authors have declared that no competing interests exist.

Synthetic lethality triggered by PARP inhibitor (PARPi) yields promising therapeutic results. Unfortunately, tumor cells acquire PARPi resistance, which is usually associated with the restoration of homologous recombination, loss of PARP1 expression, and/or loss of DNA double-strand break (DSB) end resection regulation. Here, we identify a constitutive mechanism of resistance to PARPi. We report that the bone marrow microenvironment (BMM) facilitates DSB repair activity in leukemia cells to protect them against PARPi-mediated synthetic lethality. This effect depends on the hypoxia-induced overexpression of transforming growth factor beta receptor (TGFβR) kinase on malignant cells, which is activated by bone marrow stromal cells-derived transforming growth factor beta 1 (TGF-β1). Genetic and/or pharmacological targeting of the TGF-β1-TGFβR kinase axis results in the restoration of the sensitivity of malignant cells to PARPi in BMM and prolongs the survival of leukemia-bearing mice. Our finding may lead to the therapeutic application of the TGFβR inhibitor in patients receiving PARPis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Late relapse [>3 years from complete remission (CR)] in acute lymphoblastic leukaemia (ALL), is unusual. Data from the MRC UKALLXII/ECOG E2993 trial are presented to evaluate the incidence and characteristics of late relapse in adult ALL. Of 1,909 patients, 1,752 (92%) achieved CR and among these 757 (43·2%) relapsed; 691 (91·3%) within three years and 66 (8·7%) beyond. Among these 66 patients, median time to relapse was 47 (37-144) months. Relapse beyond three years occurred in 3·8% of all who achieved CR. The cumulative risk of relapse was 40%, 43% and 45% at three, five and ten years respectively. Out of the 1 752 patients who achieved CR, 11·7% underwent autologous and 40·6% allogeneic transplant, while in CR1. Of the autologous patients, 43·2% relapsed early and 3·4% relapsed late. However, among the allogeneic patients, 13·2% relapsed early and only 1·3% late. The five-year overall survival from relapse was 5·8% and 20% in the early and late relapse patients respectively. In conclusion, late relapse in adults with ALL is not uncommon, and is associated with better outcome after relapse compared to early relapse., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Identification of genomic and epigenomic determinants of drug resistance provides important insights for improving cancer treatment. Using agnostic genome-wide interrogation of mRNA and miRNA expression, DNA methylation, SNPs, CNAs and SNVs/Indels in primary human acute lymphoblastic leukemia cells, we identified 463 genomic features associated with glucocorticoid resistance. Gene-level aggregation identified 118 overlapping genes, 15 of which were confirmed by genome-wide CRISPR screen. Collectively, this identified 30 of 38 (79%) known glucocorticoid-resistance genes/miRNAs and all 38 known resistance pathways, while revealing 14 genes not previously associated with glucocorticoid-resistance. Single cell RNAseq and network-based transcriptomic modelling corroborated the top previously undiscovered gene, CELSR2. Manipulation of CELSR2 recapitulated glucocorticoid resistance in human leukemia cell lines and revealed a synergistic drug combination (prednisolone and venetoclax) that mitigated resistance in mouse xenograft models. These findings illustrate the power of an integrative genomic strategy for elucidating genes and pathways conferring drug resistance in cancer cells., Competing Interests: Competing Interests The authors declare no competing interests.

Disruption of epigenetic regulation is a hallmark of acute myeloid leukemia (AML), but epigenetic therapy is complicated by the complexity of the epigenome. Herein, we developed a long-term primary AML ex vivo platform to determine whether targeting different epigenetic layers with 5-azacytidine and LSD1 inhibitors would yield improved efficacy. This combination was most effective in TET2 mut AML, where it extinguished leukemia stem cells and particularly induced genes with both LSD1-bound enhancers and cytosine-methylated promoters. Functional studies indicated that derepression of genes such as GATA2 contributes to drug efficacy. Mechanistically, combination therapy increased enhancer-promoter looping and chromatin-activating marks at the GATA2 locus. CRISPRi of the LSD1-bound enhancer in patient-derived TET2 mut AML was associated with dampening of therapeutic GATA2 induction. TET2 knockdown in human hematopoietic stem/progenitor cells induced loss of enhancer 5-hydroxymethylation and facilitated LSD1-mediated enhancer inactivation. Our data provide a basis for rational targeting of cooperating aberrant promoter and enhancer epigenetic marks driven by mutant epigenetic modifiers. SIGNIFICANCE: Somatic mutations of genes encoding epigenetic modifiers are a hallmark of AML and potentially disrupt many components of the epigenome. Our study targets two different epigenetic layers at promoters and enhancers that cooperate to aberrant gene silencing, downstream of the actions of a mutant epigenetic regulator. This article is highlighted in the In This Issue feature, p. 813 ., (©2019 American Association for Cancer Research.)

To improve the outcome of relapsed/refractory acute myeloid leukemia (AML), a randomized phase II trial of three novel regimens was conducted. Ninety patients were enrolled and were in first relapse or were refractory to induction/re-induction chemotherapy. They were randomized to the following regimens: carboplatin-topotecan (CT), each by continuous infusion for 5 days; alvocidib (formerly flavopiridol), cytarabine, and mitoxantrone (FLAM) in a timed sequential regimen; or sirolimus combined with mitoxantrone, etoposide, and cytarabine (S-MEC). The primary objective was attainment of a complete remission (CR). A Simon two-stage design was used for each of the three arms. The median age of the patients in the FLAM arm was older at 62 years compared with 55 years for the CT arm and the S-MEC arm. The overall response was 14% in the CT arm (5/35, 90% CI 7%-35%), 28% in the FLAM arm (10/36, 90% CI, 16%-43%), and 16% in the S-MEC arm (3/19, 90% CI, 4%-36%). There were nine treatment-related deaths, seven of which occurred in the FLAM arm with four of these in elderly patients. We conclude that the FLAM regimen had an encouraging response rate and should be considered for further clinical development but should be used with caution in elderly patients., (© 2018 Wiley Periodicals, Inc.)

Patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) may benefit from salvage chemoimmunotherapy (CIT). To explore further the use of CIT in the pre-novel agent era, ECOG-ACRIN undertook a phase 2 trial (E2903) for R/R CLL utilizing pentostatin, cyclophosphamide, and rituximab (PCR) followed by a consolidation course of alemtuzumab. This trial enrolled 102 patients with a median age of 64 years. Treatment consisted of 6 cycles of PCR followed by alemtuzumab for either 4 or 18 weeks depending on the initial response to PCR. The overall response after PCR (complete remission, CR, nodular partial remission, nPR, and partial remission, PR) was 55%. Major responses (CR or nPR) were achieved in 6%. The median overall survival (OS) and the median progression-free survival were 28 and 12 months, respectively. The most serious nonlethal adverse events were myelosuppression, febrile neutropenia, fatigue, nausea, and hyponatremia. PCR is an effective and well-tolerated nucleoside-based regimen for heavily pretreated CLL patients with R/R disease. The addition of alemtuzumab to CLL patients with a minor response (PR) or stable disease did not result in a significant number of higher responses (CR or nPR) nor an improvement in OS., (© 2019 S. Karger AG, Basel.)

This study examines the long-term OS of relapsed AML patients who were enrolled to 9 successive ECOG-ACRIN trials for newly diagnosed AML, during 1984-2008. The objectives were to examine whether there is a trend of improvement in the survival of relapsed AML patients in the more recent studies and to search for prognostic factors that are associated with long-term OS after relapse. A total of 3012 patients were enrolled, 1779 (59.1%) achieved CR1 and of these, 58.9% relapsed. The median follow-up was 9.7 years. The median OS from relapse was 0.5 years and the 5-year OS was 10 (±1)%. These results were similar even for the most recent studies. A multivariate model showed that age, cytogenetics at diagnosis, duration of CR1 and undergoing allogeneic transplantation were significantly associated with OS from relapse. Even among patients who relapsed with better prognostic factors; age < 40 and CR1 > 12 months, there was no significant OS difference between the studies. In conclusion, this large cohort appears to confirm that the survival of AML patients postrelapse continues to be dismal and has not improved during the past quarter of a century., (© 2018 Wiley Periodicals, Inc.)

In acute myeloid leukemia (AML), chemotherapy resistance remains prevalent and poorly understood. Using functional proteomics of patient AML specimens, we identified MEF2C S222 phosphorylation as a specific marker of primary chemoresistance. We found that Mef2c S222A/S222A knock-in mutant mice engineered to block MEF2C phosphorylation exhibited normal hematopoiesis, but were resistant to leukemogenesis induced by MLL-AF9 MEF2C phosphorylation was required for leukemia stem cell maintenance and induced by MARK kinases in cells. Treatment with the selective MARK/SIK inhibitor MRT199665 caused apoptosis and conferred chemosensitivity in MEF2C-activated human AML cell lines and primary patient specimens, but not those lacking MEF2C phosphorylation. These findings identify kinase-dependent dysregulation of transcription factor control as a determinant of therapy response in AML, with immediate potential for improved diagnosis and therapy for this disease. Significance: Functional proteomics identifies phosphorylation of MEF2C in the majority of primary chemotherapy-resistant AML. Kinase-dependent dysregulation of this transcription factor confers susceptibility to MARK/SIK kinase inhibition in preclinical models, substantiating its clinical investigation for improved diagnosis and therapy of AML. Cancer Discov; 8(4); 478-97. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 371 ., (©2018 American Association for Cancer Research.)

The presence of a monosomal karyotype (MK+) and/or a complex karyotype (CK+) identifies subcategories of AML with poor prognosis. The prognostic significance of the most common monosomies (monosomy 5, monosomy 7, and monosomy 17) within MK+/CK+AML is not well defined. We analyzed data from 1,592 AML patients age 17-93 years enrolled on ECOG-ACRIN therapeutic trials. The majority of MK+ patients (182/195; 93%) were MK+/CK+ with 87% (158/182) having ≥5 clonal abnormalities (CK≥5). MK+ patients with karyotype complexity ≤4 had a median overall survival (OS) of 0.4y compared to 1.0y for MK- with complexity ≤4 (p<0.001), whereas no OS difference was seen in MK+vs. MK- patients with CK≥5 (p=0.82). Monosomy 5 (93%; 50/54) typically occurred within a highly complex karyotype and had no impact on OS (0.4y; p=0.95). Monosomy 7 demonstrated no impact on OS in patients with CK≥5 (p=0.39) or CK≤4 (p=0.44). Monosomy 17 appeared in 43% (68/158) of CK≥5 patients and demonstrated statistically significant worse OS (0.4y) compared to CK≥5 patients without monosomy 17 (0.5y; p=0.012). Our data suggest that the prognostic impact of MK+is limited to those with less complex karyotypes and that monosomy 17 may independently predict for worse survival in patients with AML., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although the majority of patients with acute myeloid leukemia (AML) initially respond to chemotherapy, many of them subsequently relapse, and the mechanistic basis for AML persistence following chemotherapy has not been determined. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3A R882 ), have been observed in AML and in individuals with clonal hematopoiesis in the absence of leukemic transformation. Patients with DNMT3A R882 AML have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy, suggesting that DNMT3A R882 cells persist and drive relapse. We found that Dnmt3a mutations induced hematopoietic stem cell expansion, cooperated with mutations in the FMS-like tyrosine kinase 3 gene (Flt3 ITD ) and the nucleophosmin gene (Npm1 c ) to induce AML in vivo, and promoted resistance to anthracycline chemotherapy. In patients with AML, the presence of DNMT3A R882 mutations predicts minimal residual disease, underscoring their role in AML chemoresistance. DNMT3A R882 cells showed impaired nucleosome eviction and chromatin remodeling in response to anthracycline treatment, which resulted from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect led to an inability to sense and repair DNA torsional stress, which resulted in increased mutagenesis. Our findings identify a crucial role for DNMT3A R882 mutations in driving AML chemoresistance and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy.

Purpose Extramedullary disease (EMD) at diagnosis in patients with acute myeloid leukemia (AML) has been recognized for decades. Reported herein are results from a large study of patients with AML who were treated in consecutive ECOG-ACRIN Cancer Research Group frontline clinical trials in an attempt to define the incidence and clinical implications of EMD. Methods Patients with newly diagnosed AML, age 15 years and older, who were treated in 11 clinical trials, were studied to identify EMD, as defined by physical examination, laboratory findings, and imaging results. Results Of the 3,522 patients enrolled, 282 were excluded, including patients with acute promyelocytic leukemia, incorrect diagnosis, or no adequate assessment of EMD at baseline. The overall incidence of EMD was 23.7%. The sites involved were: lymph nodes (11.5%), spleen (7.3%), liver (5.3%), skin (4.5%), gingiva (4.4%), and CNS (1.1%). Most patients (65.3%) had only one site of EMD, 20.9% had two sites, 9.5% had three sites, and 3.4% had four sites. The median overall survival was 1.035 years. In univariable analysis, the presence of any EMD ( P = .005), skin involvement ( P = .002), spleen ( P < .001), and liver ( P < .001), but not CNS ( P = .34), nodal involvement ( P = .94), and gingival hypertrophy ( P = .24), was associated with a shorter overall survival. In contrast, in multivariable analysis, adjusted for known prognostic factors such as cytogenetic risk and WBC count, neither the presence of EMD nor the number of specific sites of EMD were independently prognostic. Conclusion This large study demonstrates that EMD at any site is common but is not an independent prognostic factor. Treatment decisions for patients with EMD should be made on the basis of recognized AML prognostic factors, irrespective of the presence of EMD., Competing Interests: Authors’ disclosures of potential conflicts of interest are found in the article online at www.jco.org. Author contributions are found at the end of this article.

Background: Variable response to chemotherapy in acute myeloid leukemia (AML) represents a major treatment challenge. Clinical and genetic features incompletely predict outcome. The value of clinical epigenetic assays for risk classification has not been extensively explored. We assess the prognostic implications of a clinical assay for multilocus DNA methylation on adult patients with de novo AML., Methods: We performed multilocus DNA methylation assessment using xMELP on samples and calculated a methylation statistic (M-score) for 166 patients from UPENN with de novo AML who received induction chemotherapy. The association of M-score with complete remission (CR) and overall survival (OS) was evaluated. The optimal M-score cut-point for identifying groups with differing survival was used to define a binary M-score classifier. This classifier was validated in an independent cohort of 383 patients from the Eastern Cooperative Oncology Group Trial 1900 (E1900; NCT00049517)., Results: A higher mean M-score was associated with death and failure to achieve CR. Multivariable analysis confirmed that a higher M-score was associated with death ( P = 0.011) and failure to achieve CR ( P = 0.034). Median survival was 26.6 months versus 10.6 months for low and high M-score groups. The ability of the M-score to perform as a classifier was confirmed in patients ≤ 60 years with intermediate cytogenetics and patients who achieved CR, as well as in the E1900 validation cohort., Conclusion: The M-score represents a valid binary prognostic classifier for patients with de novo AML. The xMELP assay and associated M-score can be used for prognosis and should be further investigated for clinical decision making in AML patients.

The initial report of the Eastern Cooperative Oncology Group-American College of Radiology Imaging Network Cancer Research Group trial E1900 (#NCT00049517) showed that induction therapy with high-dose (HD) daunorubicin (90 mg/m(2)) improved overall survival in adults <60 years old with acute myeloid leukemia (AML); however, at initial analysis, the benefit was restricted to younger patients (<50 years) and patients without unfavorable cytogenetics or aFLT3-ITD mutation. Here, we update the results of E1900 after longer follow-up (median, 80.1 months among survivors), focusing on the benefit of HD daunorubicin on common genetic subgroups. Compared with standard-dose daunorubicin (45 mg/m(2)), HD daunorubicin is associated with a hazard ratio (HR) for death of 0.74 (P= .001). Younger patients (<50 years) benefited from HD daunorubicin (HR, 0.66;P= .002), as did patients with favorable and intermediate cytogenetics (HR, 0.51;P= .03 and HR, 0.68;P= .01, respectively). Patients with unfavorable cytogenetics were shown to benefit from HD daunorubicin on multivariable analysis (adjusted HR, 0.66;P= .04). Patients with FLT3-ITD (24%),DNMT3A(24%), and NPM1(26%) mutant AML all benefited from HD daunorubicin (HR, 0.61,P= .009; HR, 0.62,P= .02; and HR, 0.50,P= .002; respectively). HD benefit was seen in the subgroup of older patients (50-60 years) with the FLT3-ITD or NPM1 mutation. Additionally, the presence of an NPM1 mutation confers a favorable prognosis only for patients receiving anthracycline dose intensification during induction., (© 2016 by The American Society of Hematology.)

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL in vivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Telomeres are the capping ends of chromosomes that protect the loss of genetic material and prevent chromosomal instability. In human tissue-specific stem/progenitor cells, telomere length (TL) is maintained by the telomerase complex, which consists of a reverse transcriptase catalytic subunit (TERT) and an RNA template (TERC). Very short telomeres and loss-of-function mutations in the TERT and TERC genes have been reported in acute myeloid leukemia, but the role of telomeres in acute promyelocytic leukemia (APL) has not been well established. We report the results for a large cohort of 187 PML/RARα-positive APL patients. No germline mutations in the TERT or TERC genes were identified. Codon 279 and 1062 TERT polymorphisms were present at a frequency similar to that in the general population. TL measured in blood or marrow mononuclear cells at diagnosis was significantly shorter in the APL patients than in healthy volunteers, and shorter telomeres at diagnosis were significantly associated with high-risk disease. For patients who achieved complete remission, the median increase in TL from diagnosis to remission (delta TL) was 2.0 kilobase (kb), and we found delta TL to be the most powerful predictor of overall survival when compared with well-established risk factors for poor outcomes in APL., Competing Interests: of Conflicts of Interest: Authors report no conflicts of interest., (© 2016 S. Karger AG, Basel.)

Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fludarabine (F) and cyclophosphamide (C) remain backbones of up-front chemotherapy regimens for chronic lymphocytic leukemia (CLL). We report long-term follow-up of a randomized F vs. FC trial in untreated CLL (#) . With median follow-up of 88 months, estimated median progression-free survival (PFS) was 19.3 vs. 48.1 months for F (n = 109) and FC (n = 118), respectively (p < 0.0001), and median overall survival (OS) was 88.0 vs. 79.1 months (p = 0.96). In multivariable analyses, variables associated with inferior PFS and OS respectively were age (p = 0.002, p < 0.001), Rai stage (p = 0.006, p = 0.02) and sex (p = 0.03, PFS only). Del(17)(p13.1) predicted shorter PFS and OS (p < 0.0001 for each), as did del(11q)(22.3) (p < 0.0001, p = 0.005, respectively), trisomy 12 with mutated Notch1 (p = 0.003, p = 0.03, respectively) and unmutated IGHV (p = 0.009, p = 0.002, respectively), all relative to patients without these features. These data confirm results from shorter follow-up and further justify targeted therapies for CLL.

Many tumors are believed to be maintained by a small number of cancer stem-like cells, where cure is thought to require eradication of this cell population. In this study, we investigated the dynamics of acute promyelocytic leukemia (APL) before and during therapy with regard to disease initiation, progression, and therapeutic response. This investigation used a mathematical model of hematopoiesis and a dataset derived from the North American Intergroup Study INT0129. The known phenotypic constraints of APL could be explained by a combination of differentiation blockade of PML-RARα-positive cells and suppression of normal hematopoiesis. All-trans retinoic acid (ATRA) neutralizes the differentiation block and decreases the proliferation rate of leukemic stem cells in vivo. Prolonged ATRA treatment after chemotherapy can cure patients with APL by eliminating the stem-like cell population over the course of approximately one year. To our knowledge, this study offers the first estimate of the average duration of therapy that is required to eliminate stem-like cancer cells from a human tumor, with the potential for the refinement of treatment strategies to better manage human malignancy., (©2014 American Association for Cancer Research.)

Rituximab, bortezomib, modified hyper-cyclophosphamide, doxorubicin, vincristine, dexamethasone (VcR-CVAD) induction chemoimmunotherapy and maintenance rituximab (MR) were evaluated for efficacy and safety in Eastern Cooperative Oncology Group protocol E1405. Patients with previously untreated mantle cell lymphoma received VcR-CVAD chemotherapy every 21 days for 6 cycles, followed by MR for 2 years. Transplant-eligible patients had the option of autologous stem cell transplantation (ASCT) consolidation instead of MR. The primary end point was the complete response (CR) rate to VcR-CVAD. The secondary end points were overall response rate (ORR), progression-free survival (PFS), overall survival (OS), and toxicities. Seventy-five eligible patients with a median age of 62 (range 40-76) were enrolled. The ORR was 95% and a CR was achieved in 68% of patients. After a median follow-up of 4.5 years, 3-year PFS and OS were 72% and 88%, respectively. No substantial difference in PFS or OS was observed between patients treated with MR (n = 44) vs ASCT (n = 22). There were no unexpected toxicities. VcR-CVAD produced high ORR and CR rates in mantle cell lymphoma. MR after VcR-CVAD induction performed similarly to ASCT and may improve response duration. Randomized clinical trials comparing MR against ASCT should be considered and randomized clinical trials evaluating bortezomib's contribution to conventional therapy are under way. This study was registered at www.clinicaltrials.gov as #NCT00433537.

Chemotherapy-related myeloid neoplasia (t-MN) is a significant late toxicity concern after cancer therapy. In the randomized intergroup phase 3 E2997 trial, initial therapy of chronic lymphocytic leukemia with fludarabine plus cyclophosphamide (FC) compared with fludarabine alone yielded higher complete and overall response rates and longer progression-free, but not overall, survival. Here, we report t-MN incidence in 278 patients enrolled in E2997 with a median 6.4-year follow-up. Thirteen cases (4.7%) of t-MN occurred at a median of 5 years from initial therapy for chronic lymphocytic leukemia, 9 after FC and 4 after fludarabine alone. By cumulative incidence methodology, rates of t-MN at 7 years were 8.2% after FC and 4.6% after fludarabine alone (P = .09). Seven of the 9 cases of t-MN after FC occurred without additional therapy. Abnormalities involving chromosomes 5 or 7 were found in 10 cases, which suggests alkylator involvement. These data suggest that FC may induce more t-MN than fludarabine alone.

We report the results of a prospective, randomized phase 3 trial evaluating the use of gemtuzumab ozogamicin (GO) in an intensive consolidation approach in 657 patients 17-60 years of age. Patients in first complete remission (CR1) after cytarabine and standard- or high-dose daunorubicin induction received 2 cycles of consolidation with high-dose cytarabine followed by peripheral blood progenitor cell collection. The 352 patients who entered consolidation were randomized to receive GO (n = 132) or not (n = 138) and then proceeded to autologous hematopoietic cell transplantation (HCT). GO was given to 67 patients. Median follow-up was 50.9 months. Results of the intention-to-treat analysis demonstrated a 4-year disease-free survival (DFS) of 33.6% versus 35.9% (P = .54) and an overall survival (OS) of 41.3% versus 41.9% (P = .52) for those randomized to receive GO versus no GO, respectively. Patients with favorable- and intermediate-risk acute myeloid leukemia (AML) treated with high-dose daunorubicin and autologous HCT had 4-year DFS rates of 60% and 40% and OS rates of 80% and 49.3%, respectively. For younger AML patients in CR1, autologous HCT should be considered in favorable- and intermediate-cytogenetic risk patients who do not have an allogeneic donor. The addition of a single dose of GO in this setting did not improve outcomes.

Zosuquidar, which modulates P-glycoprotein (P-gp) with minimal delay of anthracycline clearance, may reverse P-gp-mediated resistance in acute myeloid leukemia without increased toxicity. A total of 449 adults older than 60 years with acute myeloid leukemia or high-risk myelodysplastic syndrome enrolled in a randomized placebo-controlled double-blind trial (Eastern Cooperative Oncology Group 3999). Overall survival was compared between patients receiving conventional-dose cytarabine and daunorubicin and either zosuquidar (550 mg; 212 patients) or placebo (221 patients). Median and 2-year overall survival values were 7.2 months and 20% on zosuquidar and 9.4 months and 23% on placebo, respectively (P = .281). Remission rate was 51.9% on zosuquidar and 48.9% on placebo. All cause mortality to day 42 was not different (zosuquidar 22.2% vs placebo 16.3%; P = .158). In vitro modulation of P-gp activity by zosuquidar and expression of P-gp, multidrug resistance-related protein 1, lung resistance protein, and breast cancer resistance protein, were comparable in the 2 arms. Poor-risk cytogenetics were more common in P-gp(+) patients. P-gp expression and cytogenetics were correlated, though independent prognostic factors. We conclude that zosuquidar did not improve outcome in older acute myeloid leukemia, in part, because of the presence P-gp independent mechanisms of resistance. This trial is registered at www.clinicaltrials.gov as #NCT00046930.

The treatment of relapsed acute myeloid leukaemia (AML) remains unsatisfactory. We conducted a phase II randomized trial where patients received intermediate-dose cytarabine for 4 d followed by gemtuzumab ozogamicin on day 5 (Arm A), or combined with liposomal daunorubicin for 3 d (Arm B), or cytarabine given for 5 d combined with cyclophosphamide for 3 d and topotecan by continuous infusion for 5 d (Arm C). Eligible patients had primary refractory AML, a first relapse after a remission of <1 year, or a second or greater relapse. The primary objective of this trial was attainment of a conventional complete remission (CR) or a CR without platelet recovery (CRp) in at least 40% of patients. The CR/CRp rates for the 82 eligible patients were 3/26 (12%) in Arm A, 2/29 (7%) in Arm B, and 1/27 (4%) in Arm C. No patients who had relapsed within 6 months of initial CR or who had suffered multiple relapses responded. More than 95% of patients subsequently died of AML. No unexpected toxicities were encountered. We conclude that none of these three regimens were effective enough in the treatment of high-risk relapsed or refractory AML to warrant further study. This trial was registered at http://www.clinicaltrials.gov as #NCT00005962.

The biology and outcome of adult T-cell acute lymphoblastic leukemia are poorly understood. We present here the clinical and biologic features of 356 patients treated uniformly on the prospective trial (UKALL XII/ECOG 2993) with the aim of describing the outcome and identifying prognostic factors. Complete remission was obtained in 94% of patients, and 48% survived 5 years. Positivity of blasts for CD1a and lack of expression of CD13 were associated with better survival (P = .01 and < .001, respectively). NOTCH1 and CDKN2A mutations were seen in 61% and 42% of those tested. Complex cytogenetic abnormalities were associated with poorer survival (19% vs 51% at 5 years, P = .006). Central nervous system involvement at diagnosis did not affect survival (47% vs 48%, P = not significant). For 99 patients randomized between autograft and chemotherapy, 5-year survival was 51% in each arm. Patients with a matched sibling donor had superior 5-year survival to those without donors (61% vs 46%, chi(2), P = .02); this was the result of less relapse (25% vs 51% at 5 years, P < .001). Only 8 of 123 relapsed patients survive. This study provides a baseline for trials of new drugs, such as nelarabine, and may allow risk-adapted therapy in patients with poor-prognosis T-cell ALL.

Background: In young adults with acute myeloid leukemia (AML), intensification of the anthracycline dose during induction therapy has improved the rate of complete remission but not of overall survival. We evaluated the use of cytarabine plus either standard-dose or high-dose daunorubicin as induction therapy, followed by intensive consolidation therapy, in inducing complete remission to improve overall survival., Methods: In this phase 3 randomized trial, we assigned 657 patients between the ages of 17 and 60 years who had untreated AML to receive three once-daily doses of daunorubicin at either the standard dose (45 mg per square meter of body-surface area) or a high dose (90 mg per square meter), combined with seven daily doses of cytarabine (100 mg per square meter) by continuous intravenous infusion. Patients who had a complete remission were offered either allogeneic hematopoietic stem-cell transplantation or high-dose cytarabine, with or without a single dose of the monoclonal antibody gemtuzumab ozogamicin, followed by autologous stem-cell transplantation. The primary end point was overall survival., Results: In the intention-to-treat analysis, high-dose daunorubicin, as compared with a standard dose of the drug, resulted in a higher rate of complete remission (70.6% vs. 57.3%, P<0.001) and improved overall survival (median, 23.7 vs. 15.7 months; P=0.003). The rates of serious adverse events were similar in the two groups. Median follow-up was 25.2 months., Conclusions: In young adults with AML, intensifying induction therapy with a high daily dose of daunorubicin improved the rate of complete remission and the duration of overall survival, as compared with the standard dose. (ClinicalTrials.gov number, NCT00049517.), (2009 Massachusetts Medical Society)

Purpose: The combination of fludarabine and cyclophosphamide is an effective regimen for patients with chronic lymphocytic leukemia (CLL). However, it may be accompanied by increased toxicity compared with fludarabine alone. E2997 is a phase III randomized Intergroup trial comparing fludarabine and cyclophosphamide (FC arm) versus fludarabine (F arm) alone in patients receiving their first chemotherapy regimen for CLL., Patients and Methods: Symptomatic, previously untreated patients with CLL were randomly assigned to receive either fludarabine 25 mg/m2 intravenously (IV) days 1 through 5 or cyclophosphamide 600 mg/m2 IV day 1 and fludarabine 20 mg/m2 IV days 1 through 5. These cycles were repeated every 28 days for a maximum of six cycles., Results: A total of 278 patients were randomly assigned in this Intergroup study. Treatment with fludarabine and cyclophosphamide was associated with a significantly higher complete response (CR) rate (23.4% v 4.6%; P < .001) and a higher overall response (OR) rate (74.3% v 59.5%, P = .013) than treatment with fludarabine as a single agent. Progression-free survival (PFS) was also superior in patients treated with fludarabine and cyclophosphamide than those treated with fludarabine (31.6 v 19.2 months, P < .0001). Fludarabine and cyclophosphamide caused additional hematologic toxicity, including more severe thrombocytopenia (P = .046), but it did not increase the number of severe infections (P = .812)., Conclusion: Fludarabine and cyclophosphamide produced an increase in OR and CR, and it improved PFS in patients with previously untreated CLL compared with fludarabine alone and was not associated with an increase in infectious toxicity.