bims-tremyl 2021-01-03 papers

bims-tremyl

Biomed News

on Therapy resistance biology in myeloid leukemia

Issue of 2021‒01‒03
27 papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

Synergy of FLT3 inhibitors and the small molecule inhibitor of LIM kinase1/2 CEL_Amide in FLT3-ITD mutated Acute Myeloblastic Leukemia (AML) cells.
Targeting MCL-1 dysregulates cell metabolism and leukemia-stroma interactions and resensitizes acute myeloid leukemia to BCL-2 inhibition.
Genome-wide CRISPR screen identifies regulators of MAPK and MTOR pathways mediating sorafenib resistance in acute myeloid leukemia.
The propriety of upgrading responses to venetoclax + azacitidine in newly diagnosed patients with acute myeloid leukemia.
Megakaryocytic Expansion in Gilteritinib-Treated Acute Myeloid Leukemia Patients Is Associated With AXL Inhibition.
AML Prognoses Better with Menin-MLL Inhibitor?
Oral Azacitidine Maintenance Therapy for Acute Myeloid Leukemia in First Remission.
Cohesin mutations alter DNA damage repair and chromatin structure and create therapeutic vulnerabilities in MDS/AML.
A new cytokine-based dynamic stratification during induction is highly predictive of survivals in acute myeloid leukemia.
Using mathematical models to improve risk-scoring in acute myeloid leukemia.
OMIP 072: A 15-color panel for immunophenotypic identification, quantification, and characterization of leukemic stem cells in children with acute myeloid leukemia.
High Fructose Drives the Serine Synthesis Pathway in Acute Myeloid Leukemic Cells.
miR-21-5p promotes cell proliferation by targeting BCL11B in Thp-1 cells.
Safety profile and impact on survival of tyrosine kinase inhibitors versus conventional therapy in relapse or refractory FLT3 positive acute myeloid leukemia patients.
CD38 knockout natural killer cells expressing an affinity optimized CD38 chimeric antigen receptor successfully target acute myeloid leukemia with reduced effector cell fratricide.
Impact of gemtuzumab ozogamicin on MRD and relapse risk in patients with NPM1-mutated AML: results from the AMLSG 09-09 trial.
Immune Dysregulation and Recurring Mutations in Myelodysplastic Syndromes Pathogenesis.
Epigenetic changes in human model KMT2A leukemias highlight early events during leukemogenesis.
USP15 Deubiquitinase Safeguards Hematopoiesis and Genome Integrity in Hematopoietic Stem Cells and Leukemia Cells.
An iPSC line derived from a human acute myeloid leukemia cell line (HL-60-iPSC) retains leukemic abnormalities and displays myeloid differentiation defects.
Overexpression of CD200 and CD123 is a major influential factor in the clinical course of pediatric acute myeloid leukemia.
Acute Myeloid Leukemia Stem Cells: The Challenges of Phenotypic Heterogeneity.
Transduction of Primary AML Cells with Lentiviral Vector for In Vitro Study or In Vivo Engraftment.
Definition of a small core transcriptional circuit regulated by AML1-ETO.
Functional Dependency Analysis Identifies Potential Druggable Targets in Acute Myeloid Leukemia.
Genetic biomarkers identify a subgroup of high-risk patients within low-risk NPM1-mutated acute myeloid leukemia.
Defining the Optimal Total Number of Chemotherapy Courses in Younger Patients With Acute Myeloid Leukemia: A Comparison of Three Versus Four Courses.

Leuk Res. 2020 Dec 13. pii: S0145-2126(20)30195-8. [Epub ahead of print]100 106490

Synergy of FLT3 inhibitors and the small molecule inhibitor of LIM kinase1/2 CEL_Amide in FLT3-ITD mutated Acute Myeloblastic Leukemia (AML) cells.

Hanane Djamai, Jeannig Berrou, Mélanie Dupont, Anna Kaci, Jan Erik Ehlert, Holger Weber, André Baruchel, Fabrice Paublant, Renaud Prudent, Claude Gardin, Hervé Dombret, Thorsten Braun.

  Patients with FLT3-ITD mutated (FLT3-ITD+) Acute Myeloid Leukemia (AML), have frequently relapsed or refractory disease and FLT3-ITD+ inhibitors have limited efficacy. Rho kinases (ROCK) are constitutively activated by FLT3-ITD+ in AML via PI3 kinase and Rho GTPase. Upon activation by ROCK, LIM kinases (LIMK) inactivate cofilin by phosphorylation which affects cytoskeleton dynamics, cell growth and apoptosis. LIMK inhibition leads to cofilin activation via dephosphorylation and activated cofilin localizes to mitochondria inducing apoptosis. Thus, we investigated the therapeutic potential of the LIMK1/2 inhibitor CEL_Amide (LIMKi) in FLT3-ITD+ AML. Expression of LIMK1/2 in FLT3-ITD+ cell lines MOLM-13 and MV-4-11 cells could be detected by RT-qPCR and at the protein level. IC50 after LIMKi monotherapy was 440 nM in MOLM-13 cells and 420 nM in MV4-11 cells. Treatment with LIMKi decreased LIMK1 protein levels and repression of inactivating phosphorylation of cofilin in FLT3-ITD+ cells. Combination experiments with LIMKi and FLT3 inhibitors including midostaurin, crenolanib and gilteritinib were synergistic for treatment of MOLM-13 cells while combinations with quizartinib were additive. Combinations of LIMKi and the hypomethylating agent azacitidine or the ROCK inhibitor fasudil were additive. In NOD-SCID mice engrafted with MOLM13-LUC cells, the FLT3 inhibitor midostaurin and LIMKi delayed MOLM13-LUC engraftment as detected by in vivo bioluminescence imaging and the LIMKi and midostaurin combination prolonged significantly survival of leukemic mice. LIMK1/2 inhibition by the small molecule CEL_Amide seems to have promising activity in combination with FLT3 inhibitors in vitro as well as in vivo and may constitute a novel treatment strategy for FLT3-ITD+ AML.

Keywords:  AML; FLT3 inhibitors; FLT3-ITD; LIM kinase; LIM kinase inhibitors; ROCK

DOI:  https://doi.org/10.1016/j.leukres.2020.106490
Haematologica. 2020 Dec 23. Online ahead of print

Targeting MCL-1 dysregulates cell metabolism and leukemia-stroma interactions and resensitizes acute myeloid leukemia to BCL-2 inhibition.

Bing Z Carter, Po Yee Mak, Wenjing Tao, Marc Warmoes, Philip L Lorenzi, Duncan Mak, Vivian Ruvolo, Lin Tan, Justin Cidado, Lisa Drew, Michael Andreeff.

MCL-1 and BCL-2 are both frequently overexpressed in acute myeloid leukemia and critical for the survival of acute myeloid leukemia cells and acute myeloid leukemia stem cells. MCL-1 is a key factor in venetoclax resistance. Using genetic and pharmacological approaches, we discovered that MCL-1 regulates leukemia cell bioenergetics and carbohydrate metabolisms, including the TCA cycle, glycolysis and pentose phosphate pathway and modulates cell adhesion proteins and leukemia-stromal interactions. Inhibition of MCL-1 sensitizes to BCL-2 inhibition in acute myeloid leukemia cells and acute myeloid leukemia stem/progenitor cells, including those with intrinsic and acquired resistance to venetoclax through cooperative release of pro-apoptotic BIM, BAX, and BAK from binding to anti-apoptotic BCL-2 proteins and inhibition of cell metabolism and key stromal microenvironmental mechanisms. The combined inhibition of MCL-1 by MCL-1 inhibitor AZD5991 or CDK9 inhibitor AZD4573 and BCL-2 by venetoclax greatly extended survival of mice bearing patient-derived xenografts established from an acute myeloid leukemia patient who acquired resistance to venetoclax/decitabine. These results demonstrate that co-targeting MCL-1 and BCL-2 improves the efficacy of and overcomes preexisting and acquired resistance to BCL-2 inhibition. Activation of metabolomic pathways and leukemia-stroma interactions are newly discovered functions of MCL-1 in acute myeloid leukemia, which are independent from canonical regulation of apoptosis by MCL-1. Our data provide new mechanisms of synergy and rationale for co-targeting MCL-1 and BCL-2 clinically in patients with acute myeloid leukemia and potentially other cancers.

DOI: https://doi.org/10.3324/haematol.2020.260331
Haematologica. 2020 Dec 30. Online ahead of print

Genome-wide CRISPR screen identifies regulators of MAPK and MTOR pathways mediating sorafenib resistance in acute myeloid leukemia.

Alisa Damnernsawad, Daniel Bottomly, Stephen E Kurtz, Christopher A Eide, Shannon K McWeeney, Jeffrey W Tyner, Tamilla Nechiporuk.

Drug resistance impedes the long-term effect of targeted therapies in acute myeloid leukemia (AML), necessitating the identification of mechanisms underlying resistance. Approximately 25% of AML patients carry FLT3 mutations and develop post-treatment insensitivity to FLT3 inhibitors, including sorafenib. Using a genome-wide CRISPR screen, we identified LZTR1, NF1, TSC1 or TSC2, negative regulators of the MAPK and MTOR pathways, as mediators of sorafenib resistance. Analyses of ex vivo drug sensitivity assays in FLT3-ITD AML patient samples revealed lower expression of LZTR1, NF1, and TSC2 correlated with sorafenib sensitivity. Importantly, MAPK and/or MTOR complex1 (MTORC1) activity were upregulated in AML cells made resistant to several FLT3 inhibitors, including crenolanib, quizartinib, or sorafenib. These cells were sensitive to MEK inhibitors, and the combination of FLT3 and MEK inhibitors showed enhanced efficacy, suggesting its effectiveness in AML patients with FLT3 mutations and those with resistance to FLT3 inhibitors.

DOI: https://doi.org/10.3324/haematol.2020.257964
Leuk Lymphoma. 2020 Dec 30. 1-9

The propriety of upgrading responses to venetoclax + azacitidine in newly diagnosed patients with acute myeloid leukemia.

Diana Abbott, Evan Cherry, Maria Amaya, Christine McMahon, Marc Schwartz, Amanda Winters, Jeffrey Schowinsky, Craig T Jordan, Clayton Smith, Jonathan A Gutman, Daniel A Pollyea.

  Widely-used response criteria, conditional upon count recovery, were developed for acute myeloid leukemia (AML) in the context of intensive chemotherapy (IC). Extending these definitions to continuously-administered venetoclax-based therapies might underestimate responses. Best practices for venetoclax-based therapies mandate interruption after an end-of-cycle 1 bone marrow biopsy shows morphologic remission with cytopenias. We analyzed 435 patients with newly-diagnosed AML and follow-up response assessments. Of the 101 who responded to venetoclax + azacitidine, overall survival for patients whose response was upgraded due to count recovery during a 14-day post-disease assessment period, from complete remission (CR) with incomplete recovery of blood counts to CR, was not different compared to patients who did not need the 14-day period for count recovery. These results were distinct from 138 IC patients. Although sample sizes for the comparison groups were small, and conclusions are exploratory and must be verified, these findings support consideration of new response criteria for venetoclax-based regimens.

Keywords:  AML; ELN; response; upgrade; venetoclax

DOI:  https://doi.org/10.1080/10428194.2020.1864358
Front Oncol. 2020 ;10 585151

Megakaryocytic Expansion in Gilteritinib-Treated Acute Myeloid Leukemia Patients Is Associated With AXL Inhibition.

Kran Suknuntha, Yoon Jung Choi, Ho Sun Jung, Aditi Majumder, Sujal Shah, Igor Slukvin, Erik A Ranheim.

  Numerous recurrent genetic mutations are known to occur in acute myeloid leukemia (AML). Among these common mutations, Fms-like tyrosine kinase 3 remains as one of the most frequently mutated genes in AML. We observed apparent marrow expansion of megakaryocytes in three out of six patients with Flt3-mutated AML following treatment with a recently FDA-approved Flt3 inhibitor, gilteritinib which possesses activity against internal tandem duplication and tyrosine kinase domain Flt3 mutations and also inhibits tyrosine kinase AXL. To assess whether biopsy findings can be attributed to promotion of megakaryocytic (Mk) differentiation with gilteritinib, we devised a cellular assay by overexpressing double mutated Flt3-ITDY591F/Y919F in chronic myeloid leukemia cell line K562 to study Mk differentiation in the presence of Flt3 and AXL inhibitors with non-mutually exclusive mechanisms. These experiments demonstrated the lack of direct effect Flt3 inhibitors gilteritinib and quizartinib on megakaryocytic differentiation at either transcriptional or phenotypic levels, and highlighted antileukemic effects of AXL receptor tyrosine kinase inhibitor and its potential role in megakaryocytic development.

Keywords:  AML—acute myeloid leukemia; AXL; Flt3; gilteritinib (Xospata); internal tandem duplication (ITD); megakaryocyte; midostaurin

DOI:  https://doi.org/10.3389/fonc.2020.585151
Cancer Discov. 2020 Dec 30.

AML Prognoses Better with Menin-MLL Inhibitor?

The investigational menin-MLL inhibitor KO-539 may be active in patients with acute myeloid leukemia: In a phase I trial, the agent induced complete remissions in two patients with relapsed/refractory disease and showed signs of activity in several others.

DOI: https://doi.org/10.1158/2159-8290.CD-NB2020-120
N Engl J Med. 2020 12 24. 383(26): 2526-2537

Oral Azacitidine Maintenance Therapy for Acute Myeloid Leukemia in First Remission.

Andrew H Wei, Hartmut Döhner, Christopher Pocock, Pau Montesinos, Boris Afanasyev, Hervé Dombret, Farhad Ravandi, Hamid Sayar, Jun-Ho Jang, Kimmo Porkka, Dominik Selleslag, Irwindeep Sandhu, Mehmet Turgut, Valentina Giai, Yishai Ofran, Merih Kizil Çakar, Aida Botelho de Sousa, Justyna Rybka, Chiara Frairia, Lorenza Borin, Germana Beltrami, Jaroslav Čermák, Gert J Ossenkoppele, Ignazia La Torre, Barry Skikne, Keshava Kumar, Qian Dong, C L Beach, Gail J Roboz, .

BACKGROUND: Although induction chemotherapy results in remission in many older patients with acute myeloid leukemia (AML), relapse is common and overall survival is poor.METHODS: We conducted a phase 3, randomized, double-blind, placebo-controlled trial of the oral formulation of azacitidine (CC-486, a hypomethylating agent that is not bioequivalent to injectable azacitidine), as maintenance therapy in patients with AML who were in first remission after intensive chemotherapy. Patients who were 55 years of age or older, were in complete remission with or without complete blood count recovery, and were not candidates for hematopoietic stem-cell transplantation were randomly assigned to receive CC-486 (300 mg) or placebo once daily for 14 days per 28-day cycle. The primary end point was overall survival. Secondary end points included relapse-free survival and health-related quality of life.
RESULTS: A total of 472 patients underwent randomization; 238 were assigned to the CC-486 group and 234 were assigned to the placebo group. The median age was 68 years (range, 55 to 86). Median overall survival from the time of randomization was significantly longer with CC-486 than with placebo (24.7 months and 14.8 months, respectively; P<0.001). Median relapse-free survival was also significantly longer with CC-486 than with placebo (10.2 months and 4.8 months, respectively; P<0.001). Benefits of CC-486 with respect to overall and relapse-free survival were shown in most subgroups defined according to baseline characteristics. The most common adverse events in both groups were grade 1 or 2 gastrointestinal events. Common grade 3 or 4 adverse events were neutropenia (in 41% of patients in the CC-486 group and 24% of patients in the placebo group) and thrombocytopenia (in 22% and 21%, respectively). Overall health-related quality of life was preserved during CC-486 treatment.
CONCLUSIONS: CC-486 maintenance therapy was associated with significantly longer overall and relapse-free survival than placebo among older patients with AML who were in remission after chemotherapy. Side effects were mainly gastrointestinal symptoms and neutropenia. Quality-of-life measures were maintained throughout treatment. (Supported by Celgene; QUAZAR AML-001 ClinicalTrials.gov number, NCT01757535.).

DOI: https://doi.org/10.1056/NEJMoa2004444
JCI Insight. 2020 Dec 22. pii: 142149. [Epub ahead of print]

Cohesin mutations alter DNA damage repair and chromatin structure and create therapeutic vulnerabilities in MDS/AML.

Zuzana Tothova, Anne-Laure Valton, Rebecca Gorelov, Mounica Vallurupalli, John M Krill-Burger, Amie Holmes, Catherine C Landers, J Erika Haydu, Edyta Malolepsza, Christina R Hartigan, Melanie Donahue, Katerina D Popova, Sebastian H J Koochaki, Sergey V Venev, Jeanne F Rivera, Edwin Chen, Kasper Lage, Monica Schenone, Alan D D'Andrea, Steven A Carr, Elizabeth A Morgan, Job Dekker, Benjamin L Ebert.

  The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer including myelodysplatic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of STAG2-mutant AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to PARP inhibition. We developed a mouse model of MDS in which Stag2 mutations arise as clonal secondary lesions in the background of clonal hematopoiesis driven by Tet2 mutations, and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which is associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and RPA proteins. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies.

Keywords:  Epigenetics; Hematology; Leukemias; Mouse models; Oncology

DOI:  https://doi.org/10.1172/jci.insight.142149
Cancer Med. 2020 Dec 25.

A new cytokine-based dynamic stratification during induction is highly predictive of survivals in acute myeloid leukemia.

Pierre Peterlin, Joelle Gaschet, Thierry Guillaume, Alice Garnier, Marion Eveillard, Amandine Le Bourgeois, Michel Cherel, Camille Debord, Yannick Le Bris, Olivier Theisen, Catherine Godon, Béatrice Mahé, Viviane Dubruille, Soraya Wuilleme, Cyrille Touzeau, Thomas Gastinne, Nicolas Blin, Anne Lok, Benoît Tessoulin, Steven Le Gouill, Philippe Moreau, Marie-C Béné, Patrice Chevallier.

  The aim of this study was to assess the potential impact of the kinetics of serum levels of seven cytokines during induction in acute myeloid leukemia (AML) patients. Indeed, the role of cytokines, in the pathophysiology and response to therapy of AML patients, remains under investigation. Here, we report on the impact of peripheral levels of two cytokines, the Fms-like tyrosine kinase 3 ligand (FL) and interleukin-6 (IL-6), evaluated during first-line intensive induction. A new risk stratification can be proposed, which supersedes the ELN 2017 classification to predict survivals in AML patients by examining the kinetic profile of these cytokines during the induction phase. It segregates three groups of, respectively, high-risk, characterized by a stagnation of low FL levels, intermediate risk, with dynamic increasing FL levels and high IL-6 at day 22, and favorable risk with increasing FL levels but low IL-6 at day 22.

Keywords:  FLT3 ligand; IL-6; acute myeloid leukemia; prognostic biomarker

DOI:  https://doi.org/10.1002/cam4.3648
Chaos. 2020 Dec;30(12): 123150

Using mathematical models to improve risk-scoring in acute myeloid leukemia.

Thomas Stiehl.

Acute myeloid leukemia (AML) is an aggressive cancer of the blood forming (hematopoietic) system. Due to the high patient variability of disease dynamics, risk-scoring is an important part of its clinical management. AML is characterized by impaired blood cell formation and the accumulation of so-called leukemic blasts in the bone marrow of patients. Recently, it has been proposed to use counts of blood-producing (hematopoietic) stem cells (HSCs) as a biomarker for patient prognosis. In this work, we use a non-linear mathematical model to provide mechanistic evidence for the suitability of HSC counts as a prognostic marker. Using model analysis and computer simulations, we compare different risk-scores involving HSC quantification. We propose and validate a simple approach to improve risk prediction based on HSC and blast counts measured at the time of diagnosis.

DOI: https://doi.org/10.1063/5.0023830
Cytometry A. 2020 Dec 26.

OMIP 072: A 15-color panel for immunophenotypic identification, quantification, and characterization of leukemic stem cells in children with acute myeloid leukemia.

Marianne A Petersen, Marie Bill, Carina A Rosenberg.

  This panel was designed to identify, quantify and phenotypically characterize putative leukemic stem cells (LSCs) in bone marrow (BM) samples from individual pediatric patients diagnosed with acute myeloid leukemia (AML). Based on an aberrant expression on immunophenotypically defined hematopoietic stem cells (HSCs), several antigens have been proposed as LSC markers in AML research, using healthy adult BM samples as reference material. Generally, these antigens have been evaluated individually in smaller panels (e.g. 8-color panels). This necessitates several tubes to characterize the LSC phenotype and compromises the ability to evaluate LSC heterogeneity. The present 15-color OMIP incorporates nine suggested LSC markers to comprehensively capture LSC immunophenotypes and to explore heterogenic marker-patterns within LSC populations in a single tube. Importantly, this single tube approach requires less input material, which is essential when sampling BM aspirates from pediatric patients where sample volumes often are sparse. As knowledge on normal expression levels of the included LSC markers in HSCs from hematologically healthy children are a prerequisite for labelling a phenotype as abnormal, we have evaluated the applicability of the panel on cryopreserved mononuclear cells (MNCs) isolated from BM samples from pediatric patients without hematological disorders as well as pediatric AML patients. The panel is optimized for cryopreserved BM MNCs, but could in principle, be utilized for LSC detection in any biological material containing human hematopoietic cells.

Keywords:  aberrant marker expression; hematopoietic stem cells; human hematopoietic bone marrow; leukemic stem cells; pediatric acute myeloid leukemia; phenotypic heterogeneity

DOI:  https://doi.org/10.1002/cyto.a.24284
Cell Metab. 2020 Dec 17. pii: S1550-4131(20)30660-4. [Epub ahead of print]

High Fructose Drives the Serine Synthesis Pathway in Acute Myeloid Leukemic Cells.

Sangmoo Jeong, Angela Maria Savino, Rachel Chirayil, Ersilia Barin, Yuanming Cheng, Sun-Mi Park, Alexandra Schurer, Edouard Mullarky, Lewis C Cantley, Michael G Kharas, Kayvan R Keshari.

  A significant increase in dietary fructose consumption has been implicated as a potential driver of cancer. Metabolic adaptation of cancer cells to utilize fructose confers advantages for their malignant growth, but compelling therapeutic targets have not been identified. Here, we show that fructose metabolism of leukemic cells can be inhibited by targeting the de novo serine synthesis pathway (SSP). Leukemic cells, unlike their normal counterparts, become significantly dependent on the SSP in fructose-rich conditions as compared to glucose-rich conditions. This metabolic program is mediated by the ratio of redox cofactors, NAD+/NADH, and the increased SSP flux is beneficial for generating alpha-ketoglutarate from glutamine, which allows leukemic cells to proliferate even in the absence of glucose. Inhibition of PHGDH, a rate-limiting enzyme in the SSP, dramatically reduces leukemia engraftment in mice in the presence of high fructose, confirming the essential role of the SSP in the metabolic plasticity of leukemic cells.

Keywords:  in vivo isotope tracing; metabolic flux; redox; serine synthesis pathway

DOI:  https://doi.org/10.1016/j.cmet.2020.12.005
Oncol Lett. 2021 Feb;21(2): 119

miR-21-5p promotes cell proliferation by targeting BCL11B in Thp-1 cells.

Liang Zhang, Li Yu, Yiran Liu, Shasha Wang, Zhenfeng Hou, Jun Zhou.

  Acute myeloid leukemia (AML) is a highly heterogeneous disease that remains untreatable. MicroRNAs (miRNAs or miRs) play important roles in the pathogenesis of leukemia. miR-21 is highly expressed in multiple types of human cancer and displays oncogenic activities; however, the clinical significance of miR-21 in AML remains unclear. In the present study, it was demonstrated that miR-21 levels were high in patients with AML and in AML cell lines. Further experiments demonstrated that overexpression of miR-21 in Thp-1 human monocytes derived from acute mononuclear leukemia peripheral blood promoted cell proliferation, while downregulation of miR-21-5p, a mature sequence derived from the 5' end of the miR-21 stem-loop precursor (another mature sequence, miR-21-3p, is derived form 3' end of miR-21), inhibited cell proliferation. Specifically, it was observed that overexpression of miR-21 could promote the transition of Thp-1 cells into the S and G2/M phases of the cell cycle, as shown by flow cytometry. Furthermore, inhibition of miR-21-5p arrested cells in the S and G2/M phases. Finally, BCL11B was determined to be a functional target of miR-21-5p by luciferase assays. Our study revealed functional and mechanistic associations between miR-21 and BCL11B in Thp-1 cells, which could serve to guide clinical treatment of AML.

Keywords:  BCL11B; Thp-1 monocytes; acute myeloid leukemia; miR-21-5p

DOI:  https://doi.org/10.3892/ol.2020.12380
Leuk Res. 2020 Dec 25. pii: S0145-2126(20)30202-2. [Epub ahead of print]101 106497

Safety profile and impact on survival of tyrosine kinase inhibitors versus conventional therapy in relapse or refractory FLT3 positive acute myeloid leukemia patients.

Giovanni Marconi, Stefano De Polo, Giovanni Martinelli, Jacopo Nanni, Luca Bertamini, Annalisa Talami, Matteo Olivi, Simone Ragaini, Maria Chiara Abbenante, Chiara Sartor, Emanuela Ottaviani, Maria Teresa Bochicchio, Sarah Parisi, Maria Chiara Fontana, Gianluca Cristiano, Maddalena Raffini, Carmen Baldazzi, Nicoletta Testoni, Francesca Bonifazi, Stefania Paolini, Antonio Curti, Michele Cavo, Cristina Papayannidis.

  Relapsed or refractory (R/R) acute myeloid leukemia (AML) has a poor prognosis, and new therapies are a major clinical need. When mutated, FLT3 drives neoplastic cell proliferation. New drugs (i.e., tyrosine kinase inhibitors, TKIs) showed effectiveness in FLT3-AML and promise to change disease history and outcome. We evaluated the benefit conferred by TKIs in terms of survival, burden of complications and surrogate endpoint of quality of life in a retrospective cohort of 49 FLT3 positive, R/R AML patients. Patients who received TKIs were compared to those treated with conventional chemotherapy. Treatment with TKIs conferred a better OS and wea associated with a lower burden and severity of adverse events. Importantly, patients who received TKIs showed reduced time of hospitalization. In conclusion, treatment with TKI in R/R FLT3-AML was related to a better survival, less and milder AEs, and shorter hospitalization.

Keywords:  AML; Chemotherapy; FLT3; Safety; Survival; TKI

DOI:  https://doi.org/10.1016/j.leukres.2020.106497
Haematologica. 2020 Dec 30. Online ahead of print

CD38 knockout natural killer cells expressing an affinity optimized CD38 chimeric antigen receptor successfully target acute myeloid leukemia with reduced effector cell fratricide.

Mark Gurney, Arwen Stikvoort, Emma Nolan, Lucy Kirkham-McCarthy, Stanislav Khoruzhenko, Rama Shivakumar, Sonja Zweegman, Niels W C J Van de Donk, Tuna Mutis, Eva Szegezdi, Subhashis Sarkar, Michael O'Dwyer.

There is a strong biological rationale for the augmentation of allogeneic natural killer (NK) cell therapies with a chimeric antigen receptor (CAR) to enhance acute myeloid leukemia (AML) targeting. CD38 is an established immunotherapeutic target in multiple myeloma and under investigation as a target antigen in AML. CD38 expression on NK cells and its further induction during ex vivo NK cell expansion represents a barrier to the development of a CD38 CAR-NK cell therapy. We set out to develop a CD38 CAR-NK cell therapy for AML, first by using an NK cell line which has low baseline CD38 expression and subsequently healthy donor expanded NK cells. To overcome anticipated fratricide due to NK cell CD38 expression when using primary expanded NK cells, we applied CRISPR/Cas9 genome editing to disrupt the CD38 gene during expansion achieving a mean knockdown efficiency of 84%. The resulting CD38 KD expanded NK cells, after expression of an affinity optimized CD38 CAR, showed reduced NK cell fratricide and an enhanced ability to target primary AML blasts. Furthermore, the cytotoxic potential of CD38 CAR-NK cells was augmented by pre-treatment of the AML cells with all-trans retinoic acid which drove enhanced CD38 expression offering a rational combination therapy. These findings support the further investigation of CD38 KD - CD38 CAR-NK cells as a viable immunotherapeutic approach to the treatment of AML.

DOI: https://doi.org/10.3324/haematol.2020.271908
Blood. 2020 Dec 24. 136(26): 3041-3050

Impact of gemtuzumab ozogamicin on MRD and relapse risk in patients with NPM1-mutated AML: results from the AMLSG 09-09 trial.

Silke Kapp-Schwoerer, Daniela Weber, Andrea Corbacioglu, Verena I Gaidzik, Peter Paschka, Jan Krönke, Frauke Theis, Frank G Rücker, Maria-Veronica Teleanu, Ekaterina Panina, Nikolaus Jahn, Julia Herzig, Lena Kubanek, Anika Schrade, Gudrun Göhring, Walter Fiedler, Thomas Kindler, Thomas Schroeder, Karin T Mayer, Michael Lübbert, Mohammed Wattad, Katharina S Götze, Heinz A Horst, Elisabeth Koller, Gerald Wulf, Jan Schleicher, Martin Bentz, Jürgen Krauter, Lars Bullinger, Julia Krzykalla, Axel Benner, Richard F Schlenk, Felicitas Thol, Michael Heuser, Arnold Ganser, Hartmut Döhner, Konstanze Döhner.

Monitoring of measurable residual disease (MRD) provides prognostic information in patients with Nucleophosmin1-mutated (NPM1mut) acute myeloid leukemia (AML) and represents a powerful tool to evaluate treatment effects within clinical trials. We determined NPM1mut transcript levels (TLs) by quantitative reverse-transcription polymerase chain reaction and evaluated the prognostic impact of NPM1mut MRD and the effect of gemtuzumab ozogamicin (GO) on NPM1mut TLs and the cumulative incidence of relapse (CIR) in patients with NPM1mut AML enrolled in the randomized phase 3 AMLSG 09-09 trial. A total of 3733 bone marrow (BM) samples and 3793 peripheral blood (PB) samples from 469 patients were analyzed. NPM1mut TL log10 reduction ≥ 3 and achievement of MRD negativity in BM and PB were significantly associated with a lower CIR rate, after 2 treatment cycles and at end of treatment (EOT). In multivariate analyses, MRD positivity was consistently revealed to be a poor prognostic factor in BM and PB. With regard to treatment effect, the median NPM1mut TLs were significantly lower in the GO-Arm across all treatment cycles, resulting in a significantly greater proportion of patients achieving MRD negativity at EOT (56% vs 41%; P = .01). The better reduction in NPM1mut TLs after 2 treatment cycles in MRD positive patients by the addition of GO led to a significantly lower CIR rate (4-year CIR, 29.3% vs 45.7%, P = .009). In conclusion, the addition of GO to intensive chemotherapy in NPM1mut AML resulted in a significantly better reduction in NPM1mut TLs across all treatment cycles, leading to a significantly lower relapse rate.

DOI: https://doi.org/10.1182/blood.2020005998
Adv Exp Med Biol. 2021 Jan 01.

Immune Dysregulation and Recurring Mutations in Myelodysplastic Syndromes Pathogenesis.

Anacélia Matos, Silvia M M Magalhães, Michael J Rauh.

  Myelodysplastic syndromes (MDS) are clonal stem cell malignancies characterized by ineffective hematopoiesis leading to peripheral cytopenias and variable risk of progression to acute myeloid leukemia. Inflammation is associated with MDS pathogenesis. Several cytokines, reactive species of oxygen/nitrogen and growth factors are directly or indirectly involved in dysfunction of the MDS bone marrow (BM) microenvironment. Mutations in genes mainly regulating RNA splicing, DNA methylation and chromatin accessibility, transcription factors, signal transduction and the response to DNA damage contribute to ineffective hematopoiesis, genomic instability and MDS development. The inflammation-associated DNA damage in hematopoietic stem cells may also contribute to MDS development and progression with aggressive clinical characteristics. Many studies have aimed at clarifying mechanisms involved in the activity of immature myeloid cells as powerful modulators of the immune response and their correlation with aging, autoimmunity, and development of cancer. In this review, we explore recent advances and accumulating evidence uniting immune dysregulation, inflammaging and recurring mutations in the pathogenesis of MDS.

Keywords:  Bone marrow; Hematopoietic stem cells; Inflammation; Mutations; Myelodysplastic syndromes

DOI:  https://doi.org/10.1007/5584_2020_608
Haematologica. 2020 Dec 30. Online ahead of print

Epigenetic changes in human model KMT2A leukemias highlight early events during leukemogenesis.

Thomas Milan, Magalie Celton, Karine Lagacé, Élodie Roques, Safia Safa-Tahar-Henni, Eva Bresson, Anne Bergeron, Josée Hebert, Soheil Meshinchi, Sonia Cellot, Frédéric Barabé, Brian T Wilhelm.

Chromosomal translocations involving KMT2A gene are one of the most common genetic alterations found in pediatric acute myeloid leukemias (AML) although the molecular mechanisms that initiate the disease remain incompletely defined. To elucidate these initiating events we have used a human model system of AML driven by the KMT2A-MLLT3 (KM3) fusion. More specifically, we investigated changes in DNA methylation, histone modifications, and chromatin accessibility at each stage of our model system and correlated these with expression changes. We observe the development of a profound hypomethylation phenotype in the early stages of leukemic transformation after KM3 addition along with loss of expression of stem cell associated genes along with skewed expression in other genes such as S100A8/9 implicated in leukemogenesis. In addition, early increases in the expression of the lysine demethylase KDM4B was functionally linked to these expression changes as well as other key transcription factors. Remarkably, our ATAC-seq data showed that there were relatively few leukemiaspecific changes and the vast majority corresponded to open chromatin regions and transcription factor clusters previously observed in other cell types. Integration of the gene expression and epigenetic changes revealed the adenylate cyclase gene ADCY9 as an essential gene in KM3-AML, and suggest the potential for autocrine signalling through the chemokine receptor CCR1 and CCL23 ligand. Together, our results suggest that KM3 induces subtle changes in the epigenome while co-opting the normal transcriptional machinery to drive leukemogenesis.

DOI: https://doi.org/10.3324/haematol.2020.271619
Cell Rep. 2020 Dec 29. pii: S2211-1247(20)31522-9. [Epub ahead of print]33(13): 108533

USP15 Deubiquitinase Safeguards Hematopoiesis and Genome Integrity in Hematopoietic Stem Cells and Leukemia Cells.

Paul van den Berk, Cesare Lancini, Carlos Company, Michela Serresi, Maria Pilar Sanchez-Bailon, Danielle Hulsman, Colin Pritchard, Ji-Ying Song, Matthias Jürgen Schmitt, Ellen Tanger, Oliver Popp, Philipp Mertins, Ivo J Huijbers, Heinz Jacobs, Maarten van Lohuizen, Gaetano Gargiulo, Elisabetta Citterio.

  Altering ubiquitination by disruption of deubiquitinating enzymes (DUBs) affects hematopoietic stem cell (HSC) maintenance. However, comprehensive knowledge of DUB function during hematopoiesis in vivo is lacking. Here, we systematically inactivate DUBs in mouse hematopoietic progenitors using in vivo small hairpin RNA (shRNA) screens. We find that multiple DUBs may be individually required for hematopoiesis and identify ubiquitin-specific protease 15 (USP15) as essential for HSC maintenance in vitro and in transplantations and Usp15 knockout (KO) mice in vivo. USP15 is highly expressed in human hematopoietic tissues and leukemias. USP15 depletion in murine progenitors and leukemia cells impairs in vitro expansion and increases genotoxic stress. In leukemia cells, USP15 interacts with and stabilizes FUS (fused in sarcoma), a known DNA repair factor, directly linking USP15 to the DNA damage response (DDR). Our study underscores the importance of DUBs in preserving normal hematopoiesis and uncovers USP15 as a critical DUB in safeguarding genome integrity in HSCs and leukemia cells.

Keywords:  DNA damage response; FUS; HSC; RNAi; USP15; deubiquitinase; deubiquitinating enzymes; fused in sarcoma; genome integrity; hematopoietic stem cell; in vivo shRNA screen; leukemia

DOI:  https://doi.org/10.1016/j.celrep.2020.108533
Stem Cell Res. 2020 Dec;pii: S1873-5061(20)30397-4. [Epub ahead of print]49 102096

An iPSC line derived from a human acute myeloid leukemia cell line (HL-60-iPSC) retains leukemic abnormalities and displays myeloid differentiation defects.

Amanda E Yamasaki, Jane N Warshaw, Beverly L Kyalwazi, Hiroko Matsui, Kristen Jepsen, Athanasia D Panopoulos.

Cancer-derived iPSCs have provided valuable insight into oncogenesis, but human cancer cells can often be difficult to reprogram, especially in cases of complex genetic abnormalities. Here we report, to our knowledge, the first successful generation of an iPSC line from a human immortalized acute myeloid leukemia (AML) cell line, the cell line HL-60. This iPSC line retains a majority of the leukemic genotype and displays defects in myeloid differentiation, thus providing a tool for modeling and studying AML.

DOI: https://doi.org/10.1016/j.scr.2020.102096
Exp Mol Pathol. 2020 Dec 23. pii: S0014-4800(20)30874-1. [Epub ahead of print]118 104597

Overexpression of CD200 and CD123 is a major influential factor in the clinical course of pediatric acute myeloid leukemia.

Eman Z Kandeel, Youssef Madney, Dalia Negm Eldin, Nevine F Shafik.

  Acute myeloid leukemia (AML) accounts for approximately 20% of all pediatric acute leukemias. The outcome of AML is still unsatisfactory. CD123 and CD200 were demonstrated to play important roles in hematological malignancies. The aim of this study was to investigate the impact of CD200 and CD123 overexpression and the influence of both proteins on the clinical presentation and disease outcome. Bone marrow (BM) samples from 89 pediatric AML patients were obtained at presentation and after therapy. Cells from the bulk population and from the leukemia stem cell (LSC) compartment were examined by multi parametric flow cytometry. In the bulk population, CD200 was positive in 64/89 (71.9) samples, CD123 was positive in 62/89 (69.7%) samples, and dual CD200 and CD123 positivity was observed in 54/89 (60.7%) samples. CD200/CD123 expressions were observed in LSCs in 64/60 samples respectively (71.9%/67.4%), and co-expressed in 51 samples (57.3%). CD200 was overexpressed in secondary AML (p < 0.05). A multivariate analysis revealed that minimal residual disease (MRD) and lymphadenopathy were associated with CD200 overexpression. Moreover, lymphadenopathy, low platelet count, and MRD were independently associated with CD123 expression. The co-expression of CD200 and CD123 demonstrated a statistically significant relationship with unfavorable cytogenetic karyotypes and high total leucocyte count (TLC). The expression of CD200 and CD123 alone and together had an adverse impact on complete remission (CR), MRD positivity, and overall survival (OS). Cases with MRD on day 28 after induction displayed stable expression patterns of CD200 and CD123. CD200 and CD123 both had a negative influence on clinical presentation and treatment outcome, which remarkably worsened when both were concomitantly overexpressed. CD200 and CD123 can therefore be used as markers of MRD in AML and may also serve as therapeutic targets.

Keywords:  Acute myeloid leukemia; CD123; CD200; Flow cytometry; Leukemia stem cells; Pediatric leukemia

DOI:  https://doi.org/10.1016/j.yexmp.2020.104597
Cancers (Basel). 2020 Dec 12. pii: E3742. [Epub ahead of print]12(12):

Acute Myeloid Leukemia Stem Cells: The Challenges of Phenotypic Heterogeneity.

Marlon Arnone, Martina Konantz, Pauline Hanns, Anna M Paczulla Stanger, Sarah Bertels, Parimala Sonika Godavarthy, Maximilian Christopeit, Claudia Lengerke.

  Patients suffering from acute myeloid leukemia (AML) show highly heterogeneous clinical outcomes. Next to variabilities in patient-specific parameters influencing treatment decisions and outcome, this is due to differences in AML biology. In fact, different genetic drivers may transform variable cells of origin and co-exist with additional genetic lesions (e.g., as observed in clonal hematopoiesis) in a variety of leukemic (sub)clones. Moreover, AML cells are hierarchically organized and contain subpopulations of more immature cells called leukemic stem cells (LSC), which on the cellular level constitute the driver of the disease and may evolve during therapy. This genetic and hierarchical complexity results in a pronounced phenotypic variability, which is observed among AML cells of different patients as well as among the leukemic blasts of individual patients, at diagnosis and during the course of the disease. Here, we review the current knowledge on the heterogeneous landscape of AML surface markers with particular focus on those identifying LSC, and discuss why identification and targeting of this important cellular subpopulation in AML remains challenging.

Keywords:  acute myeloid leukemia; cellular heterogeneity; leukemic stem cells; markers; relapse

DOI:  https://doi.org/10.3390/cancers12123742
STAR Protoc. 2020 Dec 18. 1(3): 100163

Transduction of Primary AML Cells with Lentiviral Vector for In Vitro Study or In Vivo Engraftment.

Aaron D Schimmer, Rashim Pal Singh, Ayesh K Seneviratne, Geethu E Thomas, Neil MacLean, Rose Hurren.

  We describe a method to silence genes in primary acute myeloid leukemia cells by transducing them with shRNA in lentiviral vectors. The transduction of primary non-adherent cells is particularly challenging. The protocol will aid in performing such experiments and is particularly helpful to prepare cells for in vivo engraftment studies. Use of a special medium supplemented with cytokines preserves the viability of the leukemic stem cells and their ability to engraft the marrow of immune-deficient mice. For complete details on the use and execution of this protocol, please refer to Singh et al. (2020).

Keywords:  Cancer; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2020.100163
Mol Cell. 2020 Dec 21. pii: S1097-2765(20)30897-2. [Epub ahead of print]

Definition of a small core transcriptional circuit regulated by AML1-ETO.

Kristy R Stengel, Jacob D Ellis, Clare L Spielman, Monica L Bomber, Scott W Hiebert.

  Transcription factors regulate gene networks controlling normal hematopoiesis and are frequently deregulated in acute myeloid leukemia (AML). Critical to our understanding of the mechanism of cellular transformation by oncogenic transcription factors is the ability to define their direct gene targets. However, gene network cascades can change within minutes to hours, making it difficult to distinguish direct from secondary or compensatory transcriptional changes by traditional methodologies. To overcome this limitation, we devised cell models in which the AML1-ETO protein could be quickly degraded upon addition of a small molecule. The rapid kinetics of AML1-ETO removal, when combined with analysis of transcriptional output by nascent transcript analysis and genome-wide AML1-ETO binding by CUT&RUN, enabled the identification of direct gene targets that constitute a core AML1-ETO regulatory network. Moreover, derepression of this gene network was associated with RUNX1 DNA binding and triggered a transcription cascade ultimately resulting in myeloid differentiation.

Keywords:  AML1-ETO; PRO-seq; PROTAC; RUNX1; RUNX1T1; degron tag; myeloid leukemia; nascent transcription

DOI:  https://doi.org/10.1016/j.molcel.2020.12.005
Cancers (Basel). 2020 Dec 10. pii: E3710. [Epub ahead of print]12(12):

Functional Dependency Analysis Identifies Potential Druggable Targets in Acute Myeloid Leukemia.

Yujia Zhou, Gregory P Takacs, Jatinder K Lamba, Christopher Vulpe, Christopher R Cogle.

  Refractory disease is a major challenge in treating patients with acute myeloid leukemia (AML). Whereas the armamentarium has expanded in the past few years for treating AML, long-term survival outcomes have yet to be proven. To further expand the arsenal for treating AML, we searched for druggable gene targets in AML by analyzing screening data from a lentiviral-based genome-wide pooled CRISPR-Cas9 library and gene knockout (KO) dependency scores in 15 AML cell lines (HEL, MV411, OCIAML2, THP1, NOMO1, EOL1, KASUMI1, NB4, OCIAML3, MOLM13, TF1, U937, F36P, AML193, P31FUJ). Ninety-four gene KOs met the criteria of (A) specifically essential to AML cell survival, (B) non-essential in non-AML cells, and (C) druggable according to three-dimensional (3D) modeling or ligand-based druggability scoring. Forty-four of 94 gene-KOs (47%) had an already-approved drug match and comprised a drug development list termed "deKO." Fifty of 94 gene-KOs (53%) had no drug in development and comprised a drug discovery list termed "disKO." STRING analysis and gene ontology categorization of the disKO targets preferentially cluster in the metabolic processes of UMP biosynthesis, IMP biosynthesis, dihydrofolate metabolism, pyrimidine nucleobase biosynthesis, vitellogenesis, and regulation of T cell differentiation and hematopoiesis. Results from this study serve as a testable compendium of AML drug targets that, after validation, may be translated into new therapeutics.

Keywords:  CRISPR; acute myeloid leukemia; screening; target identification

DOI:  https://doi.org/10.3390/cancers12123710
Leuk Lymphoma. 2020 Dec 29. 1-12

Genetic biomarkers identify a subgroup of high-risk patients within low-risk NPM1-mutated acute myeloid leukemia.

Diego Carbonell, Julia Suárez-González, María Chicano, Cristina Andrés-Zayas, Miriam Díez-Díez, Gabriela Rodríguez-Macías, Paula Muñiz, Mi Kwon, Javier Anguita, José Luis Díez-Martín, Ismael Buño, Carolina Martínez-Laperche.

  Although acute myeloid leukemia (AML) with NPM1 mut /FLT3-ITDneg is a low-risk entity, its relapse rate remains high. Out of 333 AML patients, 27 were NPM1 mut, and were analyzed in greater detail in order to find associations between clinical and molecular features and cumulative incidence of relapse. Next-generation sequencing (NGS) was performed on diagnosis and remission samples using two capture-based panels. The presence of the FLT3 D835 variant at diagnosis and a qPCR value of NPM1 mut ≥0.1% after induction chemotherapy were associated with an increased probability of relapse, especially if both conditions are present together. By contrast, patients in which the main clone found at diagnosis harbored NPM1 variant had a lower risk of relapse. Nineteen of the 85 variants found at diagnosis were detected by NGS in remission. AML Subgroup with NPM1 mut/FLT3-ITDneg is a heterogeneous entity, which can be further risk-stratified based on molecular biomarkers.

Keywords:  Acute myeloid leukemia; FLT3; NPM1; genetic biomarkers; prognostic impact

DOI:  https://doi.org/10.1080/10428194.2020.1863400
J Clin Oncol. 2020 Dec 23. JCO2001170

Defining the Optimal Total Number of Chemotherapy Courses in Younger Patients With Acute Myeloid Leukemia: A Comparison of Three Versus Four Courses.

Alan K Burnett, Nigel H Russell, Robert K Hills, Stephen Knapper, Sylvie Freeman, Brian Huntly, Richard E Clark, Ian F Thomas, Lars Kjeldsen, Mary Frances McMullin, Mark Drummond, Jonathan Kell, Ruth Spearing.

PURPOSE: The optimum number of treatment courses for younger patients with acute myeloid leukemia (AML) is uncertain. The United Kingdom National Cancer Research Institute AML17 trial randomly assigned patients who were not high risk to a total of three versus four courses.PATIENTS AND METHODS: Patients received two induction courses based on daunorubicin and cytarabine (Ara-C), usually with gemtuzumab ozogamicin. Following remission, 1,017 patients were randomly assigned to a third course, MACE (amsacrine, etoposide, and Ara-C), plus a fourth course of MidAc (mitoxantrone and Ara-C) and following an amendment to one or two courses of high-dose Ara-C. Primary end points were cumulative incidence of relapse (CIR), relapse-free survival (RFS), and overall survival (OS). Outcomes were correlated with patient characteristics, mutations, cytogenetics, induction treatments, and measurable residual disease (MRD) postinduction.
RESULTS: In logrank analyses, CIR and RFS at 5 years were improved in recipients of four courses (50% v 58%: hazard ratio [HR] 0.81 [0.69-0.97], P = .02 and 43% v 36%: HR 0.83 [0.71-0.98], P = .03, respectively). While OS was not significantly better (63% v 57%: HR 0.84 [0.69-1.03], P = .09), the noninferiority of three courses to four courses was not established. The impact on relapse was only significant when the fourth course was Ara-C. In exploratory analyses, although MRD impacted survival, a fourth course had no effect in either MRD-positive or MRD-negative patients. A fourth course was beneficial in patients who lacked a mutation of FLT3 or NPM1, had < 3 mutations in other genes, or had a presenting WBC of < 10 × 109 L-1.
CONCLUSION: Although a fourth course of high-dose Ara-C reduced CIR and improved RFS, it did not result in a significant OS benefit. Subsets including those with favorable cytogenetics, those lacking a mutation of FLT3 or NPM1, or those with < 3 other mutations may derive survival benefit.

DOI: https://doi.org/10.1200/JCO.20.01170