bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2022–10–23
28 papers selected by
Paolo Gallipoli, Barts Cancer Institute, Queen Mary University of London
  1. Venetoclax and idasanutlin in relapsed/refractory AML: a non-randomized, open-label phase 1b trial.
  2. Finding consistency in classifications of myeloid neoplasms: a perspective on behalf of the International Workshop for Myelodysplastic Syndromes.
  3. An agenda to advance research in MDS: A TOP 10 Priority List from the first international workshop in MDS (iwMDS).
  4. A dynamic rRNA ribomethylome drives stemness in acute myeloid leukemia.
  5. Therapeutic resistance in acute myeloid leukemia cells is mediated by a novel ATM/mTOR pathway regulating oxidative phosphorylation.
  6. SF3B1 mutated MDS: Blast count, genetic co-abnormalities and their impact on classification and prognosis.
  7. HSF1 is a driver of leukemia stem cell self-renewal in acute myeloid leukemia.
  8. Molecular profiling of patients with cytogenetically normal acute myeloid leukemia and hyperleukocytosis.
  9. SF3B1 mutations in AML are strongly associated with MECOM rearrangements and may be indicative of an MDS pre-phase.
  10. Detection of TP53 Mutation in Acute Myeloid Leukemia by RT-PCR-Based Sanger Sequencing.
  11. A molecular switch between mammalian MLL complexes dictates response to Menin-MLL inhibition.
  12. An 8-year pragmatic observation evaluation of the benefits of allogeneic HCT in older and medically infirm AML patients.
  13. Molecular predictors of immunophenotypic measurable residual disease clearance in acute myeloid leukemia.
  14. Association of hematologic response and assay sensitivity on the prognostic impact of measurable residual disease in acute myeloid leukemia: a systematic review and meta-analysis.
  15. Srsf2P95H/+ co-operates with loss of TET2 to promote myeloid bias and initiate a chronic myelomonocytic leukemia-like disease in mice.
  16. Molecular landscape of immune pressure and escape in aplastic anemia.
  17. A novel network pharmacology approach for leukaemia differentiation therapy using Mogrify®.
  18. WT1 and DNMT3A play essential roles in the growth of certain patient AML cells in mice.
  19. Survival, response rates, and post-transplant outcomes in patients with Acute Myeloid Leukemia aged 60-75 treated with high intensity chemotherapy vs. lower intensity targeted therapy.
  20. Lenalidomide and Eltrombopag for Treatment of Low or Intermediate Risk Myelodysplastic Syndrome: Result of a Phase 2 Clinical Trial.
  21. Secondary fusion proteins as a mechanism of BCR::ABL1 kinase-independent resistance in chronic myeloid leukaemia.
  22. Haploidentical Versus Matched Sibling Donor Hematopoietic Stem Cell Transplantation for Adult Patients With Relapsed/Refractory Acute Lymphoblastic Leukemia: A Study From the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.
  23. Significance of hereditary gene alterations for the pathogenesis of adult bone marrow failure versus myeloid neoplasia.
  24. Clinical significance of mitochondrial DNA content in acute promyelocytic leukaemia.
  25. A 5-microRNA signature derived from core binding factor-AML is predictive in adult and childhood AML.
  26. METTL3 mediates chemoresistance by enhancing AML homing and engraftment via ITGA4.
  27. Trends in survival and cure after allogeneic haematopoietic cell transplantation for acute myeloid leukaemia from 2000 to 2020: A Danish population-based cohort study.
  28. Clonogenic assays improve determination of variant allele frequency of driver mutations in myeloproliferative neoplasms.
  1. Blood. 2022 Oct 20. pii: blood.2022016362. [Epub ahead of print]
    Venetoclax and idasanutlin in relapsed/refractory AML: a non-randomized, open-label phase 1b trial.
    Naval G Daver, Monique Dail, Jacqueline S Garcia, Brian A Jonas, Karen W L Yee, Kevin R Kelly, Norbert Vey, Sarit Assouline, Gail J Roboz, Stefania Paolini, Daniel A Pollyea, Agostino Tafuri, Joseph M Brandwein, Arnaud Pigneux, Bayard L Powell, Pierre Fenaux, Rebecca L Olin, Giuseppe Visani, Giovanni Martinelli, Maika Onishi, Jue Wang, Weize Huang, Cherie Green, Marion G Ott, Wan-Jen Hong, Marina Y Konopleva, Michael Andreeff.
      This phase 1b trial (NCT02670044) evaluated venetoclax-idasanutlin in patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) ineligible for cytotoxic chemotherapy. Two-dimensional dose escalation (DE, n = 50) was performed for venetoclax daily with idasanutlin on days 1-5 in 28-day cycles followed by dosing schedule optimization (n = 6) to evaluate reduced venetoclax schedules (21-/14-day dosing). Common adverse events (occurring in ≥40% of patients) included diarrhea (87.3% of patients), nausea (74.5%), vomiting (52.7%), hypokalemia (50.9%), and febrile neutropenia (45.5%). During DE, across all doses, composite complete remission (CRc; CR + CR with incomplete blood count recovery + CR with incomplete platelet count recovery) rate was 26.0% and morphologic leukemia-free state (MLFS) rate was 12%. For anticipated recommended phase 2 doses (venetoclax 600 mg + idasanutlin 150 mg; venetoclax 600 mg + idasanutlin 200 mg), combined CRc rate was 34.3% and MLFS rate was 14.3%. Pretreatment IDH1/2 and RUNX1 mutations were associated with higher CRc rates (50.0% and 45.0%, respectively). CRc rate in patients with TP53 mutations was 20.0%, with responses noted among those with co-occurring IDH and RUNX1 mutations. In 12/36 evaluable patients, 25 emergent TP53 mutations were observed; 22 were present at baseline with low TP53 variant allele frequency (median 0.0095% [range, 0.0006-0.4]). Venetoclax-idasanutlin showed manageable safety and encouraging efficacy in unfit R/R AML patients. IDH1/2 and RUNX1 mutations were associated with venetoclax-idasanutlin sensitivity, even in some patients with co-occurring TP53 mutations; most emergent TP53 clones were pre-existing. Our findings will aid ongoing/future trials of BCL-2/MDM2 inhibitor combinations.
    DOI:  https://doi.org/10.1182/blood.2022016362
  2. Leukemia. 2022 Oct 20.
    Finding consistency in classifications of myeloid neoplasms: a perspective on behalf of the International Workshop for Myelodysplastic Syndromes.
    Amer M Zeidan, Jan Philipp Bewersdorf, Rena Buckstein, Mikkael A Sekeres, David P Steensma, Uwe Platzbecker, Sanam Loghavi, Jacqueline Boultwood, Rafael Bejar, John M Bennett, Uma Borate, Andrew M Brunner, Hetty Carraway, Jane E Churpek, Naval G Daver, Matteo Della Porta, Amy E DeZern, Fabio Efficace, Pierre Fenaux, Maria E Figueroa, Peter Greenberg, Elizabeth A Griffiths, Stephanie Halene, Robert P Hasserjian, Christopher S Hourigan, Nina Kim, Tae Kon Kim, Rami S Komrokji, Vijay Kutchroo, Alan F List, Richard F Little, Ravi Majeti, Aziz Nazha, Stephen D Nimer, Olatoyosi Odenike, Eric Padron, Mrinal M Patnaik, Gail J Roboz, David A Sallman, Guillermo Sanz, Maximilian Stahl, Daniel T Starczynowski, Justin Taylor, Zhuoer Xie, Mina Xu, Michael R Savona, Andrew H Wei, Omar Abdel-Wahab, Valeria Santini.
      
    DOI:  https://doi.org/10.1038/s41375-022-01724-9
  3. Blood Adv. 2022 Oct 19. pii: bloodadvances.2022008747. [Epub ahead of print]
    An agenda to advance research in MDS: A TOP 10 Priority List from the first international workshop in MDS (iwMDS).
    Maximilian Stahl, Omar Abdel-Wahab, Andrew H Wei, Michael R Savona, Mina L Xu, Zhuoer Xie, Justin Taylor, Daniel T Starczynowski, Guillermo F Sanz, David A Sallman, Valeria Santini, Gail J Roboz, Mrinal M Patnaik, Eric Padron, Olatoyosi Odenike, Aziz Nazha, Stephen D Nimer, Ravindra Majeti, Richard F Little, Steven D Gore, Alan F List, Vijay Kuchroo, Rami S Komrokji, Tae Kon Kim, Nina Kim, Christopher S Hourigan, Robert P Hasserjian, Stephanie Halene, Elizabeth A Griffiths, Peter L Greenberg, Maria E Figueroa, Pierre Fenaux, Fabio Efficace, Amy E DeZern, Matteo G Della Porta, Naval G Daver, Jane E Churpek, Hetty E Carraway, Andrew M Brunner, Uma Borate, John M Bennett, Rafael Bejar, Jacqueline Boultwood, Sanam Loghavi, Jan Philipp Bewersdorf, Uwe Platzbecker, David P Steensma, Mikkael A Sekeres, Rena J Buckstein, Amer M Zeidan.
      
    DOI:  https://doi.org/10.1182/bloodadvances.2022008747
  4. Cancer Discov. 2022 Oct 19. pii: CD-22-0210. [Epub ahead of print]
    A dynamic rRNA ribomethylome drives stemness in acute myeloid leukemia.
    Fengbiao Zhou, Nesrine Aroua, Yi Liu, Christian Rohde, Jingdong Cheng, Anna-Katharina Wirth, Daria Fijalkowska, Stefanie Göllner, Michelle Lotze, Haiyang Yun, Xiaobing Yu, Caroline Pabst, Tim Sauer, Thomas Oellerich, Hubert Serve, Christoph Rollig, Martin Bornhauser, Christian Thiede, Claudia Baldus, Michaela Frye, Simon Raffel, Jeroen Krijgsveld, Irmela Jeremias, Roland Beckmann, Andreas Trumpp, Carsten Muller-Tidow.
      The development and regulation of malignant self-renewal remains an unresolved issue. Here, we provide biochemical, genetic, and functional evidence that dynamics in ribosomal RNA (rRNA) 2'-O-methylation regulate leukemia stem cell (LSC) activity in vivo. A comprehensive analysis of the rRNA 2'-O-methylation landscape of 94 acute myeloid leukemia (AML) patients revealed dynamic 2'-O-methylation specifically at exterior sites of ribosomes. rRNA 2'-O-methylation pattern is closely associated with AML development stage and LSC gene expression signature. Forced expression of 2'-O-methyltransferase FBL induced an AML stem cell phenotype and enabled engraftment of non-LSC leukemia cells in NSG mice. Enhanced 2'-O-methylation redirected the ribosome translation program towards amino acid transporter mRNAs enriched in optimal codons and subsequently increased intracellular amino acid levels. Methylation at the single site 18S-guanosine 1447 was instrumental for LSC activity. Collectively, our work demonstrates that dynamic 2'-O-Me at specific sites on ribosomal RNAs shifts translational preferences and controls AML-LSC self-renewal.
    DOI:  https://doi.org/10.1158/2159-8290.CD-22-0210
  5. Elife. 2022 Oct 19. pii: e79940. [Epub ahead of print]11
    Therapeutic resistance in acute myeloid leukemia cells is mediated by a novel ATM/mTOR pathway regulating oxidative phosphorylation.
    Hae J Park, Mark A Gregory, Vadym Zaberezhnyy, Andrew Goodspeed, Craig T Jordan, Jeffrey S Kieft, James DeGregori.
      While leukemic cells are susceptible to various therapeutic insults, residence in the bone marrow microenvironment typically confers protection from a wide range of drugs. Thus, understanding the unique molecular changes elicited by the marrow is of critical importance towards improving therapeutic outcomes. In the present study, we demonstrate that aberrant activation of oxidative phosphorylation serves to induce therapeutic resistance in FLT3 mutant human AML cells challenged with FLT3 inhibitor drugs. Importantly, our findings show that AML cells are protected from apoptosis following FLT3 inhibition due to marrow-mediated activation of ATM, which in turn up-regulates oxidative phosphorylation via mTOR signaling. mTOR is required for the bone marrow stroma-dependent maintenance of protein translation, with selective polysome enrichment of oxidative phosphorylation transcripts, despite FLT3 inhibition. To investigate the therapeutic significance of this finding, we tested the mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib in primary human AML xenograft models. While marrow resident AML cells were highly resistant to quizartinib alone, the addition of everolimus induced profound reduction in tumor burden and prevented relapse. Taken together, these data provide a novel mechanistic understanding of marrow-based therapeutic resistance, and a promising strategy for improved treatment of FLT3 mutant AML patients.
    Keywords:  cancer biology; human
    DOI:  https://doi.org/10.7554/eLife.79940
  6. Leukemia. 2022 Oct 19.
    SF3B1 mutated MDS: Blast count, genetic co-abnormalities and their impact on classification and prognosis.
    Sandra Huber, Torsten Haferlach, Manja Meggendorfer, Stephan Hutter, Gregor Hoermann, Constance Baer, Wolfgang Kern, Claudia Haferlach.
      Recently, MDS with mutated SF3B1 and blast count <5% was proposed as distinct entity with favorable prognosis by the international working group for the prognosis of MDS (IWG-PM), the 5th edition of the WHO classification and the International Consensus Classification. To further characterize this entity with respect to the genomic landscape, AML transformation rate and clinical outcome, we analyzed 734 MDS patients by whole genome sequencing. SF3B1 mutations were identified in 31% (n = 231), most frequently accompanied by TET2 mutations (29%). 144/231 (62%) SF3B1mut samples fulfilled entity criteria proposed by IWG-PM (SF3B1ent). These cases were associated with longer survival, lower AML transformation rate, normal karyotypes and harbored less accompanying mutations compared to SF3B1mut samples not falling into the proposed SF3B1 entity (SF3B1nent). Of SF3B1mut cases 7% (15/231; SF3B1ent: 3/144 [2%]; SF3B1nent: 12/87 [14%]) progressed to AML compared to 15% SF3B1 wild-type patients (75/503). Of these 15 SF3B1mut cases, 10 (67%) showed RUNX1 mutations at MDS or AML stage. Multivariate analysis revealed that del(5q) and RUNX1 mutations were independent negative prognostic factors for overall survival, while blast count >5% was not. In conclusion, SF3B1mut MDS has a favorable prognosis independent of blast count if karyotype and RUNX1 mutations are considered.
    DOI:  https://doi.org/10.1038/s41375-022-01728-5
  7. Nat Commun. 2022 Oct 16. 13(1): 6107
    HSF1 is a driver of leukemia stem cell self-renewal in acute myeloid leukemia.
    Qianze Dong, Yan Xiu, Yang Wang, Christina Hodgson, Nick Borcherding, Craig Jordan, Jane Buchanan, Eric Taylor, Brett Wagner, Mariah Leidinger, Carol Holman, Dennis J Thiele, Sean O'Brien, Hai-Hui Xue, Jinming Zhao, Qingchang Li, Howard Meyerson, Brendan F Boyce, Chen Zhao.
      Acute myeloid leukemia (AML) is maintained by self-renewing leukemic stem cells (LSCs). A fundamental problem in treating AML is that conventional therapy fails to eliminate LSCs, which can reinitiate leukemia. Heat shock transcription factor 1 (HSF1), a central regulator of the stress response, has emerged as an important target in cancer therapy. Using genetic Hsf1 deletion and a direct HSF1 small molecule inhibitor, we show that HSF1 is specifically required for the maintenance of AML, while sparing steady-state and stressed hematopoiesis. Mechanistically, deletion of Hsf1 dysregulates multifaceted genes involved in LSC stemness and suppresses mitochondrial oxidative phosphorylation through downregulation of succinate dehydrogenase C (SDHC), a direct HSF1 target. Forced expression of SDHC largely restores the Hsf1 ablation-induced AML developmental defect. Importantly, the growth and engraftment of human AML cells are suppressed by HSF1 inhibition. Our data provide a rationale for developing efficacious small molecules to specifically target HSF1 in AML.
    DOI:  https://doi.org/10.1038/s41467-022-33861-1
  8. Cancer. 2022 Oct 22.
    Molecular profiling of patients with cytogenetically normal acute myeloid leukemia and hyperleukocytosis.
    Friederike Pastore, Alessandro Pastore, Maja Rothenberg-Thurley, Klaus H Metzeler, Bianka Ksienzyk, Stephanie Schneider, Stefan K Bohlander, Jan Braess, Maria C Sauerland, Dennis Görlich, Wolfgang E Berdel, Bernhard Wörmann, Michael S von Bergwelt-Baildon, Wolfgang Hiddemann, Karsten Spiekermann.
       BACKGROUND: Acute myeloid leukemia (AML) with initial hyperleukocytosis is associated with high early mortality and a poor prognosis. The aims of this study were to delineate the underlying molecular landscape in the largest cytogenetic risk group, cytogenetically normal acute myeloid leukemia (CN-AML), and to assess the prognostic relevance of recurrent mutations in the context of hyperleukocytosis and clinical risk factors.
    METHODS: The authors performed a targeted sequencing of 49 recurrently mutated genes in 56 patients with newly diagnosed CN-AML and initial hyperleukocytosis of ≥100 G/L treated in the AMLCG99 study. The median number of mutated genes per patient was 5. The most common mutations occurred in FLT3 (73%), NPM1 (75%), and TET2 (45%).
    RESULTS: The predominant pathways affected by mutations were signaling (84% of patients), epigenetic modifiers (75% of patients), and nuclear transport (NPM1; 75%) of patients. AML with hyperleukocytosis was enriched for molecular subtypes that negatively affected the prognosis, including a high percentage of patients presenting with co-occurring mutations in signaling and epigenetic modifiers such as FLT3 internal tandem duplications and TET2 mutations.
    CONCLUSIONS: Despite these unique molecular features, clinical risk factors, including high white blood count, hemoglobin level, and lactate dehydrogenase level at baseline, remained the predictors for overall survival and relapse-free survival in hyperleukocytotic CN-AML.
    Keywords:  acute myeloid leukemia (AML); hyperleukocytosis; molecular profiling; normal karyotype
    DOI:  https://doi.org/10.1002/cncr.34495
  9. Leukemia. 2022 Oct 21.
    SF3B1 mutations in AML are strongly associated with MECOM rearrangements and may be indicative of an MDS pre-phase.
    Sandra Huber, Torsten Haferlach, Manja Meggendorfer, Stephan Hutter, Gregor Hoermann, Constance Baer, Wolfgang Kern, Claudia Haferlach.
      
    DOI:  https://doi.org/10.1038/s41375-022-01734-7
  10. Methods Mol Biol. 2023 ;2594 87-95
    Detection of TP53 Mutation in Acute Myeloid Leukemia by RT-PCR-Based Sanger Sequencing.
    Emily R Novak, Anagha Deshpande, Darren Finlay, James R Mason, Aniruddha J Deshpande, Peter D Adams, Sha Li.
      The TP53 gene is known to be one of the most frequently mutated genes in various human cancers. In de novo acute myeloid leukemia (AML), TP53 has been found to be mutated in ~10% of patients. Although the frequency of TP53 mutations in AML is substantially lower compared to other human cancers, TP53 mutations in AML are associated with poor response to chemotherapy and poor outcomes. Therefore, assessment of TP53 status is critical in clinical routines and research studies. In this chapter, we described the use of conventional RT-PCR for rapid detection of TP53 mutations by Sanger sequencing. We use AML cells as an example but provide sufficient details for usage in other cell types.
    Keywords:  AML; Mutation; RT-PCR; Sanger sequencing; TP53
    DOI:  https://doi.org/10.1007/978-1-0716-2815-7_7
  11. Cancer Discov. 2022 Oct 20. pii: CD-22-0416. [Epub ahead of print]
    A molecular switch between mammalian MLL complexes dictates response to Menin-MLL inhibition.
    Yadira M Soto-Feliciano, Francisco J Sanchez-Rivera, Florian Perner, Douglas W Barrows, Edward R Kastenhuber, Yu-Jui Ho, Thomas S Carroll, Yijun Xiong, Disha Anand, Alexey A Soshnev, Leah Gates, Mary Clare Beytagh, David Cheon, Shengqing Stan Gu, X Shirley Liu, Andrei V Krivtsov, Maximiliano Meneses, Elisa de Stanchina, Richard M Stone, Scott A Armstrong, Scott W Lowe, C David Allis.
      Menin interacts with oncogenic MLL1-fusion proteins, and small molecules that disrupt these associations are in clinical trials for leukemia treatment. By integrating chromatin-focused and genome-wide CRISPR screens with genetic, pharmacological, and biochemical approaches, we discovered a conserved molecular switch between the MLL1-Menin and MLL3/4-UTX chromatin modifying complexes that dictates response to Menin-MLL inhibitors. MLL1-Menin safeguards leukemia survival by impeding binding of the MLL3/4-UTX complex at a subset of target gene promoters. Disrupting the Menin-MLL1 interaction triggers UTX-dependent transcriptional activation of a tumor suppressive program that dictates therapeutic responses in murine and human leukemia. Therapeutic re-activation of this program using CDK4/6 inhibitors mitigates treatment resistance in leukemia cells that are insensitive to Menin-inhibitors. These findings shed light on novel functions of evolutionary conserved epigenetic mediators like MLL1-Menin and MLL3/4-UTX and are relevant to understand and target molecular pathways determining therapeutic responses in ongoing clinical trials.
    DOI:  https://doi.org/10.1158/2159-8290.CD-22-0416
  12. Blood. 2022 Oct 19. pii: blood.2022016916. [Epub ahead of print]
    An 8-year pragmatic observation evaluation of the benefits of allogeneic HCT in older and medically infirm AML patients.
    Mohamed Lotfy Sorror, Ted A Gooley, Barry E Storer, Aaron T Gerds, Mikkael A Sekeres, Bruno C Medeiros, Eunice S Wang, Paul J Shami, Kehinde U A Adekola, Selina M Luger, Maria R Baer, David A Rizzieri, Tanya Wildes, Jamie Koprivnikar, Julie Smith, Mitchell Garrison, Kiarash Kojouri, Tammy A Schuler, Wendy M Leisenring, Lynn Onstad, Pamela S Becker, Stephanie J Lee, Brenda M Sandmaier, Frederick R Appelbaum, Elihu Estey.
      We designed a prospective, observational study enrolling patients presenting for treatment of AML at 13 institutions to analyze associations between hematopoietic cell transplantation (HCT) and survival, quality of life (QOL), function and geriatric health, in 6 groups: 1) the entire cohort, 2) ≥65 years old, 3) high comorbidity burden, 4) intermediate cytogenetic-risk, 5) adverse cytogenetic-risk, and 6) first complete remission with or without measurable residual disease. Patient health and preferences were assessed eight times across 2 years. Time-dependent regression models were used. Among 692 evaluable patients, 46% received HCT with 2-year survival of 58%. In unadjusted models, HCT was associated with reduced risks of mortality in the entire group and most of the subgroups. However, after accounting for covariates associated with increased mortality (age, comorbidity-burden, disease risks, frailty, impaired QOL, depression, and impaired function), the associations between HCT and longer survival disappeared in all groups. While function, social life, performance status, and depressive symptoms were better for those selected for HCT compared to those who were not, these health advantages were lost after receiving HCT. Recipients and non-recipients of HCT similarly ranked and expected cure as main goal of therapy, while physicians expected more cure for the formers. Accounting for health impairments negate survival benefits from HCT for AML, suggesting that the unadjusted observed benefit is due mostly to selection of the healthier candidates. Considering patients' overall expectations of cure but also the QOL burdens of HCT motivate the need for randomized trials to identify the best candidates for HCT.
    DOI:  https://doi.org/10.1182/blood.2022016916
  13. Am J Hematol. 2022 Oct 17.
    Molecular predictors of immunophenotypic measurable residual disease clearance in acute myeloid leukemia.
    Maximilian Stahl, Andriy Derkach, Noushin Farnoud, Jan Philipp Bewersdorf, Troy Robinson, Christopher Famulare, Christina Cho, Sean Devlin, Kamal Menghrajani, Minal A Patel, Sheng F Cai, Linde A Miles, Robert L Bowman, Mark B Geyer, Andrew Dunbar, Zachary D Epstein-Peterson, Erin McGovern, Jessica Schulman, Jacob L Glass, Justin Taylor, Aaron D Viny, Eytan M Stein, Bartlomiej Getta, Maria E Arcila, Qi Gao, Juliet Barker, Brian C Shaffer, Esperanza B Papadopoulos, Boglarka Gyurkocza, Miguel-Angel Perales, Omar Abdel-Wahab, Ross L Levine, Sergio A Giralt, Yanming Zhang, Wenbin Xiao, Nidhi Pai, Elli Papaemmanuil, Martin S Tallman, Mikhail Roshal, Aaron D Goldberg.
      Measurable residual disease (MRD) is a powerful prognostic factor in acute myeloid leukemia (AML). However, pre-treatment molecular predictors of immunophenotypic MRD clearance remain unclear. We analyzed a dataset of 211 patients with pre-treatment next-generation sequencing who received induction chemotherapy and had MRD assessed by serial immunophenotypic monitoring after induction, subsequent therapy, and allogeneic stem cell transplant (allo-SCT). Induction chemotherapy led to MRD- remission, MRD+ remission, and persistent disease in 35%, 27%, and 38% of patients, respectively. With subsequent therapy, 34% of patients with MRD+ and 26% of patients with persistent disease converted to MRD-. Mutations in CEBPA, NRAS, KRAS, and NPM1 predicted high rates of MRD-, while mutations in TP53, SF3B1, ASXL1 and RUNX1 and karyotypic abnormalities including inv(3), monosomy 5 or 7 predicted low rates of MRD-. Patients with fewer individual clones were more likely to achieve MRD- remission. Among 132 patients who underwent allo-SCT, outcomes were favorable whether patients achieved early MRD- after induction or later MRD- after subsequent therapy prior to allo-SCT. As MRD conversion with chemotherapy prior to allo-SCT is rarely achieved in patients with specific baseline mutational patterns and high clone numbers, upfront inclusion of these patients into clinical trials should be considered. This article is protected by copyright. All rights reserved.
    DOI:  https://doi.org/10.1002/ajh.26757
  14. Leukemia. 2022 Oct 19.
    Association of hematologic response and assay sensitivity on the prognostic impact of measurable residual disease in acute myeloid leukemia: a systematic review and meta-analysis.
    Nicholas J Short, Chenqi Fu, Donald A Berry, Roland B Walter, Sylvie D Freeman, Christopher S Hourigan, Xuelin Huang, Graciela Nogueras Gonzalez, Hyunsoo Hwang, Xinyue Qi, Hagop Kantarjian, Shouhao Zhou, Farhad Ravandi.
      Measurable residual disease (MRD) is associated with relapse and survival in acute myeloid leukemia (AML). We aimed to quantify the impact of MRD on outcomes across clinical contexts, including its association with hematologic response and MRD assay sensitivity. We performed systematic literature review and meta-analysis of 48 studies that reported the association between MRD and overall survival (OS) or disease-free survival (DFS) in AML and provided information on the MRD threshold used and the hematologic response of the study population. Among studies limited to patients in complete remission (CR), the estimated 5-year OS for the MRD-negative and MRD-positive groups was 67% (95% Bayesian credible interval [CrI], 53-77%) and 31% (95% CrI, 18-44%), respectively. Achievement of an MRD-negative response was associated with superior DFS and OS, regardless of MRD threshold or analytic sensitivity. Among patients in CR, the benefit of MRD negativity was highest in studies using an MRD cutoff less than 0.1%. The beneficial impact of MRD negativity was observed across MRD assays and timing of MRD assessment. In patients with AML in morphological remission, achievement of MRD negativity is associated with superior DFS and OS, irrespective of hematologic response or the MRD threshold used.
    DOI:  https://doi.org/10.1038/s41375-022-01692-0
  15. Leukemia. 2022 Oct 21.
    Srsf2P95H/+ co-operates with loss of TET2 to promote myeloid bias and initiate a chronic myelomonocytic leukemia-like disease in mice.
    Jane Jialu Xu, Alistair M Chalk, Meaghan Wall, Wallace Y Langdon, Monique F Smeets, Carl R Walkley.
      Recurrent mutations in RNA splicing proteins and epigenetic regulators contribute to the development of myelodysplastic syndrome (MDS) and related myeloid neoplasms. In chronic myelomonocytic leukemia (CMML), SRSF2 mutations occur in ~50% of patients and TET2 mutations in ~60%. Clonal analysis indicates that either mutation can arise as the founder lesion. Based on human cancer genetics we crossed an inducible Srsf2P95H/+ mutant model with Tet2fl/fl mice to mutate both concomitantly in hematopoietic stem cells. At 20-24 weeks post mutation induction, we observed subtle differences in the Srsf2/Tet2 mutants compared to either single mutant. Under conditions of native hematopoiesis with aging, we see a distinct myeloid bias and monocytosis in the Srsf2/Tet2 mutants. A subset of the compound Srsf2/Tet2 mutants display an increased granulocytic and distinctive monocytic proliferation (myelomonocytic hyperplasia), with increased immature promonocytes and monoblasts and binucleate promonocytes. Exome analysis of progressed disease demonstrated mutations in genes and pathways similar to those reported in human CMML. Upon transplantation, recipients developed leukocytosis, monocytosis, and splenomegaly. We reproduce Srsf2/Tet2 co-operativity in vivo, yielding a disease with core characteristics of CMML, unlike single Srsf2 or Tet2 mutation. This model represents a significant step toward building high fidelity and genetically tractable models of CMML.
    DOI:  https://doi.org/10.1038/s41375-022-01727-6
  16. Leukemia. 2022 Oct 17.
    Molecular landscape of immune pressure and escape in aplastic anemia.
    Simona Pagliuca, Carmelo Gurnari, Colin Hercus, Sébastien Hergalant, Niroshan Nadarajah, Adam Wahida, Laila Terkawi, Minako Mori, Weiyin Zhou, Valeria Visconte, Stephen Spellman, Shahinaz M Gadalla, Caiying Zhu, Ping Zhu, Torsten Haferlach, Jaroslaw P Maciejewski.
      Idiopathic aplastic anemia (IAA) pathophysiology is dominated by autoreactivity of human leukocyte antigen (HLA)-restricted T-cells against antigens presented by hematopoietic stem and progenitor cells (HSPCs). Expansion of PIGA and HLA class I mutant HSPCs have been linked to immune evasion from T-cell mediated pressures. We hypothesized that in analogy with antitumor immunity, the pathophysiological cascade of immune escape in IAA is initiated by immunoediting pressures and culminates with mechanisms of clonal evolution characterized by hits in immune recognition and response genes. To that end, we studied the genetic and transcriptomic make-up of the antigen presentation complexes in a large cohort of patients with IAA and paroxysmal nocturnal hemoglobinuria (PNH) by using single-cell RNA, high throughput DNA sequencing and single nucleotide polymorphism (SNP)-array platforms. At disease onset, HSPCs displayed activation of selected HLA class I and II-restricted mechanisms, without extensive inhibition of immune checkpoint apparatus. Using a newly implemented bioinformatic framework we found that not only class I but also class II genes were often impaired by acquisition of genetic aberrations. We also demonstrated the presence of novel somatic alterations in immune genes possibly contributing to the evasion from the autoimmune T-cells. In contrast, these hits were absent in myeloid neoplasia. These aberrations were not mutually exclusive with PNH and did not correlate with the accumulation of myeloid-driver hits. Our findings shed light on the mechanisms of immune activation and escape in IAA and define alternative modes of clonal hematopoiesis.
    DOI:  https://doi.org/10.1038/s41375-022-01723-w
  17. Oncogene. 2022 Oct 21.
    A novel network pharmacology approach for leukaemia differentiation therapy using Mogrify®.
    Lin Ming Lee, Eleni G Christodoulou, Pavithra Shyamsunder, Bei Jun Chen, Kian Leong Lee, Tsz Kan Fung, Chi Wai Eric So, Gee Chuan Wong, Enrico Petretto, Owen J L Rackham, S Tiong Ong.
      Acute myeloid leukaemia (AML) is a rapidly fatal blood cancer that is characterised by the accumulation of immature myeloid cells in the blood and bone marrow as a result of blocked differentiation. Methods which identify master transcriptional regulators of AML subtype-specific leukaemia cell states and their combinations could be critical for discovering novel differentiation-inducing therapies. In this proof-of-concept study, we demonstrate a novel utility of the Mogrify® algorithm in identifying combinations of transcription factors (TFs) and drugs, which recapitulate granulocytic differentiation of the NB4 acute promyelocytic leukaemia (APL) cell line, using two different approaches. In the first approach, Connectivity Map (CMAP) analysis of these TFs and their target networks outperformed standard approaches, retrieving ATRA as the top hit. We identify dimaprit and mebendazole as a drug combination which induces myeloid differentiation. In the second approach, we show that genetic manipulation of specific Mogrify®-identified TFs (MYC and IRF1) leads to co-operative induction of APL differentiation, as does pharmacological targeting of these TFs using currently available compounds. We also show that loss of IRF1 blunts ATRA-mediated differentiation, and that MYC represses IRF1 expression through recruitment of PML-RARα, the driver fusion oncoprotein in APL, to the IRF1 promoter. Finally, we demonstrate that these drug combinations can also induce differentiation of primary patient-derived APL cells, and highlight the potential of targeting MYC and IRF1 in high-risk APL. Thus, these results suggest that Mogrify® could be used for drug discovery or repositioning in leukaemia differentiation therapy for other subtypes of leukaemia or cancers.
    DOI:  https://doi.org/10.1038/s41388-022-02505-5
  18. Blood. 2022 Oct 18. pii: blood.2022016411. [Epub ahead of print]
    WT1 and DNMT3A play essential roles in the growth of certain patient AML cells in mice.
    Maryam Ghalandary, Yuqiao Gao, Diana Amend, Ginte Kutkaite, Binje Vick, Karsten Spiekermann, Maja Rothenberg-Thurley, Klaus H Metzeler, Anetta Marcinek, Marion Subklewe, Michael P Menden, Vindi Jurinovic, Ehsan Bahrami, Irmela Jeremias.
      
    DOI:  https://doi.org/10.1182/blood.2022016411
  19. Front Oncol. 2022 ;12 1017194
    Survival, response rates, and post-transplant outcomes in patients with Acute Myeloid Leukemia aged 60-75 treated with high intensity chemotherapy vs. lower intensity targeted therapy.
    Kieran Sahasrabudhe, Ying Huang, Melanie Rebechi, Patrick Elder, Alice Mims, Sarah Wall.
      The United States Food and Drug Administration has approved several oral, targeted therapies for the treatment of Acute Myeloid Leukemia (AML) in recent years. These agents are approved in patients with relapsed/refractory disease or as frontline therapy in patients who are ineligible for intensive chemotherapy based on age, performance status, or comorbidities. They are also being increasingly utilized frontline in patients of all ages and fitness levels through clinical trials and off label prescribing, but comparative treatment outcomes associated with intensive versus targeted therapy have not been extensively studied. We conducted a single center, retrospective analysis to address the impact of treatment intensity on survival in patients with AML aged 60-75 at diagnosis. This study included 127 patients, 73 of whom received high intensity chemotherapy at any point during treatment (any HiC) and 54 of whom received only low intensity targeted therapy (LITT only). Overall survival (OS) from treatment initiation did not differ significantly between the any HiC and LITT only groups (hazard ratio (HR) for death, 0.67; 95% CI, 0.41 to 1.09; P=0.11). The only three variables that were independently associated with superior OS were lower European Leukemia Net (ELN) risk classification, TP53 unmutated status, and receipt of transplant. Our data suggest that baseline genomic features and receipt of transplant are more important than treatment intensity in predicting survival in this patient population. They also highlight the vital role of transplant in older patients with AML regardless of treatment intensity utilized for remission induction. Larger studies are needed to further address this question, including prospective randomized trials.
    Keywords:  Acute Myeloid Leukemia; hematopoietic stem cell transplantation; intensive chemotherapy; older adult oncology; targeted therapy
    DOI:  https://doi.org/10.3389/fonc.2022.1017194
  20. Clin Cancer Res. 2022 Oct 18. pii: CCR-22-1457. [Epub ahead of print]
    Lenalidomide and Eltrombopag for Treatment of Low or Intermediate Risk Myelodysplastic Syndrome: Result of a Phase 2 Clinical Trial.
    Jesus D Gonzalez-Lugo, Suman Kambhampati, Abdulraheem Yacoub, William B Donnellan, Jesus Berdeja, Prafulla Bhagat, Karen Fehn, Cassady Remy, Sakshi Jasra, Mohammed Kazemi, Kith Pradhan, Mimi Kim, Ioannis Mantzaris, R Alejandro Sica, Nishi Shah, Mendel Goldfinger, Noah Kornblum, Kira Gritsman, Ira Braunschweig, Ulrich Steidl, Britta Will, Aditi Shastri, Amit Verma.
       PURPOSE: Thrombocytopenia is a serious complication of MDS associated with an increased bleeding risk and worse prognosis. Eltrombopag (ELT), a thrombopoietin receptor agonist, can increase platelet counts and reverse anti-megakaryopoietic effects of lenalidomide (LEN) in preclinical studies. We hypothesized ELT would reduce the incidence of thrombocytopenia in MDS.
    EXPERIMENTAL DESIGN: We conducted a phase 2 multicenter trial of ELT and LEN in adult patients with low or intermediate-1-risk MDS with symptomatic or transfusion-dependent anemia or thrombocytopenia (NCT01772420). Thrombocytopenic patients were started on ELT and subsequently treated with LEN after platelets were increased. Patients without thrombocytopenia were started on LEN monotherapy and treated with ELT if they became thrombocytopenic.
    RESULTS: 52 patients were enrolled; mean age was 71 years (range 34-93). ORR in the ITT population was 35% (18/52). ELT monotherapy led to ORR of 33.3% (7/21), 29% achieving HI-Platelets, and 24% bilineage responses. LEN monotherapy had 38% ORR (6/16) with all responders achieving HI-Erythroid. 15 patients received both ELT and LEN with ORR of 33.3%, 20% achieved HI-Erythroid, and 20% HI-Platelets with 13% bilineage responses. Median duration of response was 40 weeks for ELT (range 8-ongoing), 41 weeks (25-ongoing) for LEN, and 88 weeks (8.3-ongoing) for ELT/LEN. Non-hematological grade 3-4 treatment-related adverse events were infrequent. Among patients on ELT, 2 had major bleeding events, 1 had a reversible increase in peripheral blasts, and 1 developed marrow fibrosis after 6 years on ELT.
    CONCLUSION/DISCUSSION: ELT and LEN are well tolerated and effective in achieving hematological improvement in patients with low/intermediate-risk MDS.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-22-1457
  21. Br J Haematol. 2022 Oct 20.
    Secondary fusion proteins as a mechanism of BCR::ABL1 kinase-independent resistance in chronic myeloid leukaemia.
    Evan J Barnes, Christopher A Eide, Andy Kaempf, Daniel Bottomly, Kyle A Romine, Beth Wilmot, Dominick Saunders, Shannon K McWeeney, Cristina E Tognon, Brian J Druker.
      Drug resistance in chronic myeloid leukaemia (CML) may occur via mutations in the causative BCR::ABL1 fusion or BCR::ABL1-independent mechanisms. We analysed 48 patients with BCR::ABL1-independent resistance for the presence of secondary fusion genes by RNA sequencing. We identified 10 of the most frequently detected secondary fusions in 21 patients. Validation studies, cell line models, gene expression analysis and drug screening revealed differences with respect to proliferation rate, differentiation and drug sensitivity. Notably, expression of RUNX1::MECOM led to resistance to ABL1 tyrosine kinase inhibitors in vitro. These results suggest secondary fusions contribute to BCR::ABL1-independent resistance and may be amenable to combined therapies.
    Keywords:  CML; chromosomal rearrangements; drug resistance
    DOI:  https://doi.org/10.1111/bjh.18515
  22. Hemasphere. 2022 Nov;6(11): e790
    Haploidentical Versus Matched Sibling Donor Hematopoietic Stem Cell Transplantation for Adult Patients With Relapsed/Refractory Acute Lymphoblastic Leukemia: A Study From the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.
    Arnon Nagler, Myriam Labopin, Ryszard Swoboda, Pietro Pioltelli, Mutlu Arat, Ibrahim Yakoub-Agha, Alexander Kulagin, Anna Maria Raiola, Hakan Ozdogu, Antonio Risitano, Zubeyde Nur Ozkurt, Jaime Sanz, Eolia Brissot, Peric Zina, Sebastian Giebel, Fabio Ciceri, Mohamad Mohty.
      The results of haploidentical stem cell transplantation (haploHCT) for patients with acute lymphoblastic leukemia (ALL) transplanted in active disease remain largely unknown. We retrospectively analyzed adult patients with R/R ALL who underwent haploHCT or matched sibling donor (MSD-HCT) as a first transplantation between 2012 and 2020. The analysis comprised 274 patients, 94 had a haploHCT, and 180 had an MSD-HCT. The median follow-up was 32 months. The median age was 33 (range 18-76) and 37 (18-76) years in the haplo- and MSD-HCT groups, respectively. Post-transplant cyclophosphamide (PTCy) was used in 88% of haploHCT and in 4% of the MSD-HCT group. Graft-versus-host disease grade III-IV was higher in haploHCT than in the MSD-HCT group (18% versus 9%; P = 0.042). The 2-year chronic (c) graft-versus-host disease rates were 17% versus 33% (hazard ratio [HR] = 0.56; P = 0.14), respectively. By multivariate analysis, relapse incidence, and leukemia-free survival were not significatively different between the transplant groups, while nonrelapse mortality (NRM) was significantly higher (25% versus 18% at 2 years; HR = 2.03; P = 0.042) and overall survival (OS) lower (22% versus 38% at 2 years; HR = 1.72; P = 0.009) in the haploHCT group compared with the MSD-HCT group. We conclude that the 2-year OS of R/R ALL patients undergoing MSD transplants is significantly better than in haploHCT with a higher NRM in the latter.
    DOI:  https://doi.org/10.1097/HS9.0000000000000790
  23. Leukemia. 2022 Oct 20.
    Significance of hereditary gene alterations for the pathogenesis of adult bone marrow failure versus myeloid neoplasia.
    Yasuo Kubota, Misam Zawit, Jibran Durrani, Wenyi Shen, Waled Bahaj, Tariq Kewan, Ben Ponvilawan, Minako Mori, Manja Meggendorfer, Carmelo Gurnari, Thomas LaFramboise, Simone Feurstein, Mikkael A Sekeres, Valeria Visconte, Lucy A Godley, Torsten Haferlach, Jaroslaw P Maciejewski.
      Broader genetic screening has led to the growing recognition of the role of germline variants associated with adult bone marrow failure (BMF) and myeloid neoplasia (MN) not exclusively in children and young adults. In this study, we applied a germline variant panel to 3008 adult BMF and MN cases to assess the importance of germline genetics and its impact on disease phenotype and prognosis. In our cohort, up to 9.7% of BMF and 5.3% of MN cases carried germline variants. Our cohort also included heterozygous carriers of recessive traits, suggesting they contribute to the risk of BMF and MN. By gene category, variants of Fanconi anemia gene family represented the highest-frequency category for both BMF and MN cases, found in 4.9% and 1.7% cases, respectively. In addition, about 1.4% of BMF and 0.19% of MN cases harbored multiple germline variants affecting often functionally related genes as compound heterozygous. The burden of germline variants in BMF and MN was clearly associated with acquisition of monosomy 7. While BMF cases carrying germline variants showed similar overall survival as compared to the wild-type (WT) cases, MN cases with germline variants experienced a significantly shorter overall survival as compared to WT cases.
    DOI:  https://doi.org/10.1038/s41375-022-01729-4
  24. Br J Haematol. 2022 Oct 20.
    Clinical significance of mitochondrial DNA content in acute promyelocytic leukaemia.
    Diego A Pereira-Martins, Juan L Coelho-Silva, Isabel Weinhäuser, Pedro L Franca-Neto, Douglas R Silveira, César Ortiz, Amanda Moreira-Aguiar, Marinus M Lima, Luisa C Koury, Raul A de Melo, Ana B Glória, Evandro M Fagundes, Bruno K Lino, Katia Pagnano, Rosane Bittencourt, Elenaide Nunes, Fabiola Traina, Lorena Figueiredo-Pontes, Armand Keating, Martin S Tallman, Raul C Ribeiro, Richard Dilon, Arnold Ganser, Miguel A Sanz, Nancy Berliner, Peter Valk, Bob Löwenberg, Tiziana Ottone, Nelida I Noguera, Maria T Voso, Francesca Paoloni, Paola Fazi, Emanuele Ammatuna, Gerwin Huls, Jan Jacob Schuringa, Eduardo M Rego, Antonio R Lucena-Araujo.
      Although a growing body of evidence demonstrates that altered mtDNA content (mtDNAc) has clinical implications in several types of solid tumours, its prognostic relevance in acute promyelocytic leukaemia (APL) patients remains largely unknown. Here, we show that patients with higher-than-normal mtDNAc had better outcomes regardless of tumour burden. These results were more evident in patients with low-risk of relapse. The multivariate Cox proportional hazard model demonstrated that high mtDNAc was independently associated with a decreased cumulative incidence of relapse. Altogether, our data highlights the possible role of mitochondrial metabolism in APL patients treated with ATRA.
    Keywords:  ATRA; anthracycline-based chemotherapy; mtDNA content; oxidative phosphorylation
    DOI:  https://doi.org/10.1111/bjh.18510
  25. Leuk Res. 2022 Oct 08. pii: S0145-2126(22)00344-7. [Epub ahead of print]123 106968
    A 5-microRNA signature derived from core binding factor-AML is predictive in adult and childhood AML.
    Martino Marco Gabra, Jonathan Tak-Sum Chow, Taehyung Kim, Meong Hi Son, Andrea Arruda, John F Woolley, Zhaolei Zhang, Mark D Minden, Leonardo Salmena.
      
    Keywords:  Core binding factor. acute myeloid leukemia; MiRNA signature; MicroRNA; Prognosis; Survival
    DOI:  https://doi.org/10.1016/j.leukres.2022.106968
  26. Leukemia. 2022 Oct 20.
    METTL3 mediates chemoresistance by enhancing AML homing and engraftment via ITGA4.
    Mingying Li, Jingjing Ye, Yuan Xia, Meng Li, Guosheng Li, Xiang Hu, Xiuhua Su, Dongmei Wang, Xin Zhao, Fei Lu, Jingxin Li, Daoxin Ma, Tao Sun, Chunyan Ji.
      Chemoresistant leukemia relapse is one of the most common causes of death for acute myeloid leukemia (AML) patients and the homing/engraftment in bone marrow (BM) are crucial steps for AML cells to acquire chemoresistance by interacting with stromal cell components. No crosstalk between m6A modification and homing/engraftment has been reported. Here, we performed comprehensive high-throughput analyses, including RNA sequencing of CR (complete remission) and relapsed AML patients, and reverse-phase protein arrays of chemoresistant cells to identify METTL3 as a key player regulating AML chemoresistance. Then, METTL3-mediated m6A modification was proved to induce the chemoresistance in vitro and in vivo. Furthermore, AML homing/engraftment was discovered being enhanced by upregulated-METTL3 in chemoresistant cells. And the homing/engraftment and drug-resistance associated phenotypes of chemoresistant cells could be reversed by a METTL3 inhibitor. Mechanistically, METTL3 extended the half-life of ITGA4 mRNA by m6A methylation, and then, increased expression of ITGA4 protein to enhance homing/engraftment of AML cells. The results provide insights into the function of m6A modification on the interaction between AML cells and BM niches and clarify the relationship between METTL3 and AML homing/engraftment, suggesting a therapeutic strategy for the treatment of refractory/relapsed AML with METTL3 inhibitors.
    DOI:  https://doi.org/10.1038/s41375-022-01696-w
  27. Br J Haematol. 2022 Oct 20.
    Trends in survival and cure after allogeneic haematopoietic cell transplantation for acute myeloid leukaemia from 2000 to 2020: A Danish population-based cohort study.
    Lars K Gjaerde, Lasse H Jakobsen, Caroline Juhl-Christensen, Gitte Olesen, Irma Petruskevicius, Marianne T Severinsen, Claus W Marcher, Kim Theilgaard-Mönch, Niels S Andersen, Lone S Friis, Brian Kornblit, Søren L Petersen, Ida Schjødt, Henrik Sengeløv.
      
    DOI:  https://doi.org/10.1111/bjh.18511
  28. Ann Hematol. 2022 Oct 21.
    Clonogenic assays improve determination of variant allele frequency of driver mutations in myeloproliferative neoplasms.
    Milena Kalmer, Kristina Pannen, Rebecca Lemanzyk, Chiara Wirths, Julian Baumeister, Angela Maurer, Kim Kricheldorf, Joelle Schifflers, Deniz Gezer, Susanne Isfort, Tim H Brümmendorf, Steffen Koschmieder, Nicolas Chatain.
      Molecular diagnostics moves more into focus as technology advances. In patients with myeloproliferative neoplasms (MPN), identification and monitoring of the driver mutations have become an integral part of diagnosis and monitoring of the disease. In some patients, none of the known driver mutations (JAK2V617F, CALR, MPL) is found, and they are termed "triple negative" (TN). Also, whole-blood variant allele frequency (VAF) of driver mutations may not adequately reflect the VAF in the stem cells driving the disease. We reasoned that colony forming unit (CFU) assay-derived clonogenic cells may be better suited than next-generation sequencing (NGS) of whole blood to detect driver mutations in TN patients and to provide a VAF of disease-driving cells. We have included 59 patients carrying the most common driver mutations in the establishment or our model. Interestingly, cloning efficiency correlated with whole blood VAF (p = 0.0048), suggesting that the number of disease-driving cells correlated with VAF. Furthermore, the clonogenic VAF correlated significantly with the NGS VAF (p < 0.0001). This correlation was lost in patients with an NGS VAF <15%. Further analysis showed that in patients with a VAF <15% by NGS, clonogenic VAF was higher than NGS VAF (p = 0.003), suggesting an enrichment of low numbers of disease-driving cells in CFU assays. However, our approach did not enhance the identification of driver mutations in 5 TN patients. A significant correlation of lactate dehydrogenase (LDH) serum levels with both CFU- and NGS-derived VAF was found. Our results demonstrate that enrichment for clonogenic cells can improve the detection of MPN driver mutations in patients with low VAF and that LDH levels correlate with VAF.
    Keywords:  CALR; Clonogenic assay; Driver mutations; JAK2V617F; Lactate dehydrogenase (LDH); MPL; Next-generation sequencing (NGS); Triple negative MPN; Variant allele frequency (VAF)
    DOI:  https://doi.org/10.1007/s00277-022-05000-9
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