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Purpose: Persistent molecular disease (PMD) after induction chemotherapy predicts relapse in AML. In this study, we used whole-exome sequencing (WES) and targeted error-corrected sequencing to assess the frequency and mutational patterns of PMD in 30 patients with AML., Materials and Methods: The study cohort included 30 patients with adult AML younger than 65 years who were uniformly treated with standard induction chemotherapy. Tumor/normal WES was performed for all patients at presentation. PMD analysis was evaluated in bone marrow samples obtained during clinicopathologic remission using repeat WES and analysis of patient-specific mutations and error-corrected sequencing of 40 recurrently mutated AML genes (MyeloSeq)., Results: WES for patient-specific mutations detected PMD in 63% of patients (19/30) using a minimum variant allele fraction (VAF) of 2.5%. In comparison, MyeloSeq identified persistent mutations above 0.1% VAF in 77% of patients (23/30). PMD was usually present at relatively high levels (>2.5% VAFs), such that WES and MyeloSeq agreed for 73% of patients despite differences in detection limits. Mutations in DNMT3A , ASXL1 , and TET2 (ie, DTA mutations) were persistent in 16 of 17 patients, but WES also detected non-DTA mutations in 14 of these patients, which for some patients distinguished residual AML cells from clonal hematopoiesis. Surprisingly, MyeloSeq detected additional variants not identified at presentation in 73% of patients that were consistent with new clonal cell populations after chemotherapy., Conclusion: PMD and clonal hematopoiesis are both common in patients with AML in first remission. These findings demonstrate the importance of baseline testing for accurate interpretation of mutation-based tumor monitoring assays for patients with AML and highlight the need for clinical trials to determine whether these complex mutation patterns correlate with clinical outcomes in AML.

Progression from myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (AML) is associated with the acquisition and expansion of subclones. Our understanding of subclone evolution during progression, including the frequency and preferred order of gene mutation acquisition, remains incomplete. Sequencing of 43 paired MDS and secondary AML samples identified at least one signaling gene mutation in 44% of MDS and 60% of secondary AML samples, often below the level of standard sequencing detection. In addition, 19% of MDS and 47% of secondary AML patients harbored more than one signaling gene mutation, almost always in separate, coexisting subclones. Signaling gene mutations demonstrated diverse patterns of clonal evolution during disease progression, including acquisition, expansion, persistence, and loss of mutations, with multiple patterns often coexisting in the same patient. Multivariate analysis revealed that MDS patients who had a signaling gene mutation had a higher risk of AML progression, potentially providing a biomarker for progression., Significance: Subclone expansion is a hallmark of progression from MDS to secondary AML. Subclonal signaling gene mutations are common at MDS (often at low levels), show complex and convergent patterns of clonal evolution, and are associated with future progression to secondary AML. See related article by Guess et al., p. 316 (33). See related commentary by Romine and van Galen, p. 270. This article is highlighted in the In This Issue feature, p. 265., (©2022 American Association for Cancer Research.)

Natural killer (NK) cells are a promising alternative to T cells for cancer immunotherapy. Adoptive therapies with allogeneic, cytokine-activated NK cells are being investigated in clinical trials. However, the optimal cytokine support after adoptive transfer to promote NK cell expansion, and persistence remains unclear. Correlative studies from 2 independent clinical trial cohorts treated with major histocompatibility complex-haploidentical NK cell therapy for relapsed/refractory acute myeloid leukemia revealed that cytokine support by systemic interleukin-15 (IL-15; N-803) resulted in reduced clinical activity, compared with IL-2. We hypothesized that the mechanism responsible was IL-15/N-803 promoting recipient CD8 T-cell activation that in turn accelerated donor NK cell rejection. This idea was supported by increased proliferating CD8+ T-cell numbers in patients treated with IL-15/N-803, compared with IL-2. Moreover, mixed lymphocyte reactions showed that IL-15/N-803 enhanced responder CD8 T-cell activation and proliferation, compared with IL-2 alone. Additionally, IL-15/N-803 accelerated the ability of responding T cells to kill stimulator-derived memory-like NK cells, demonstrating that additional IL-15 can hasten donor NK cell elimination. Thus, systemic IL-15 used to support allogeneic cell therapy may paradoxically limit their therapeutic window of opportunity and clinical activity. This study indicates that stimulating patient CD8 T-cell allo-rejection responses may critically limit allogeneic cellular therapy supported with IL-15. This trial was registered at www.clinicaltrials.gov as #NCT03050216 and #NCT01898793., (© 2022 by The American Society of Hematology.)

Leukemia stem cells utilize cell adhesion molecules like CXCR4/CXCL12 to home to bone marrow stromal niches where they are maintained in a dormant, protected state. Dociparstat sodium (DSTAT, CX-01) is a low anticoagulant heparin with multiple mechanisms of action, including inhibition of the CXCR4/CXCL12 axis, blocking HMGB1, and binding platelet factor 4 (PF-4). We conducted a pilot study adding DSTAT to azacitidine for patients with AML or MDS unresponsive to or relapsed after prior hypomethylating agent therapy, hypothesizing that DSTAT may improve response rates. Twenty patients were enrolled, with a median of 2 prior lines of therapy and 6 cycles of prior hypomethylating agents. Among fifteen patients evaluable for response, there was 1 complete remission, and 3 marrow complete remissions, for a response rate of 27 % among evaluable patients (20 % overall). Hematologic improvement was observed in 5 additional patients. The median overall survival for all enrolled patients was 205 days (95 % CI 119-302). While cytopenias and infections were common, these were not out of proportion to what would be expected in this population of patients undergoing treatment with azacitidine alone. In summary, this trial demonstrated the feasibility of combining DSTAT with azacitidine, with several responses observed, suggesting this combination warrants further study., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Genomic analysis is essential for risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Whole-genome sequencing is a potential replacement for conventional cytogenetic and sequencing approaches, but its accuracy, feasibility, and clinical utility have not been demonstrated., Methods: We used a streamlined whole-genome sequencing approach to obtain genomic profiles for 263 patients with myeloid cancers, including 235 patients who had undergone successful cytogenetic analysis. We adapted sample preparation, sequencing, and analysis to detect mutations for risk stratification using existing European Leukemia Network (ELN) guidelines and to minimize turnaround time. We analyzed the performance of whole-genome sequencing by comparing our results with findings from cytogenetic analysis and targeted sequencing., Results: Whole-genome sequencing detected all 40 recurrent translocations and 91 copy-number alterations that had been identified by cytogenetic analysis. In addition, we identified new clinically reportable genomic events in 40 of 235 patients (17.0%). Prospective sequencing of samples obtained from 117 consecutive patients was performed in a median of 5 days and provided new genetic information in 29 patients (24.8%), which changed the risk category for 19 patients (16.2%). Standard AML risk groups, as defined by sequencing results instead of cytogenetic analysis, correlated with clinical outcomes. Whole-genome sequencing was also used to stratify patients who had inconclusive results by cytogenetic analysis into risk groups in which clinical outcomes were measurably different., Conclusions: In our study, we found that whole-genome sequencing provided rapid and accurate genomic profiling in patients with AML or MDS. Such sequencing also provided a greater diagnostic yield than conventional cytogenetic analysis and more efficient risk stratification on the basis of standard risk categories. (Funded by the Siteman Cancer Research Fund and others.)., (Copyright © 2021 Massachusetts Medical Society.)

Purpose: Physicians treating hematologic malignancies increasingly order targeted sequencing panels to interrogate recurrently mutated genes. The precise impact of these panels on clinical decision making is not well understood., Methods: Here, we report our institutional experience with a targeted 40-gene panel (MyeloSeq) that is used to generate a report for both genetic variants and variant allele frequencies for the treating physician (the limit of mutation detection is approximately one AML cell in 50)., Results: In total, 346 sequencing reports were generated for 325 patients with suspected hematologic malignancies over an 8-month period (August 2018 to April 2019). To determine the influence of genomic data on clinical care for patients with acute myeloid leukemia (AML), we analyzed 122 consecutive reports from 109 patients diagnosed with AML and surveyed the treating physicians with a standardized questionnaire. The panel was ordered most commonly at diagnosis (61.5%), but was also used to assess response to therapy (22.9%) and to detect suspected relapse (15.6%). The panel was ordered at multiple timepoints during the disease course for 11% of patients. Physicians self-reported that 50 of 114 sequencing reports (44%) influenced clinical care decisions in 44 individual patients. Influences were often nuanced and extended beyond identifying actionable genetic variants with US Food and Drug Administration-approved drugs., Conclusion: This study provides insights into how physicians are currently using multigene panels capable of detecting relatively rare AML cells. The most influential way to integrate these tools into clinical practice will be to perform prospective clinical trials that assess patient outcomes in response to genomically driven interventions., Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments). Erica K. BarnellEmployment: Geneoscopy Stock and Other Ownership Interests: Geneoscopy Patents, Royalties, Other Intellectual Property: Inventor of intellectual property in start-up company (Geneoscopy) Travel, Accommodations, Expenses: GeneoscopyKenneth F. NewcomerEmployment: Geneoscopy (I) Stock and Other Ownership Interests: Geneoscopy (I) Patents, Royalties, Other Intellectual Property: Inventor of intellectual property owned by Geneoscopy (I)Zachary L. SkidmoreStock and Other Ownership Interests: Aim Immunotech, Catalyst PharmaceuticalsKilannin KrysiakConsulting or Advisory Role: Gerson Lehrman GroupLukas D. WartmanConsulting or Advisory Role: Novartis, IncyteStephen T. OhConsulting or Advisory Role: Incyte, Novartis, Blueprint Medicines, Celgene, Kartos, CTI BioPharma Corp, PharmaEssentia, Disc Medicine, Constellation Pharmaceuticals Research Funding: Incyte (Inst), Gilead Sciences (Inst), CTI BioPharma Corp (Inst), Kartos (Inst), Celgene (Inst), Sierra Oncology (Inst), Blueprint Medicines (Inst), Constellation Pharmaceuticals (Inst)John S. WelchConsulting or Advisory Role: Archer Biosciences, Agios Research Funding: Janssen Oncology (Inst), Notable Labs (Inst)Keith E. Stockerl-GoldsteinStock and Other Ownership Interests: Abbott Laboratories, AbbVie Consulting or Advisory Role: Celgene Research Funding: GlaxoSmithKline, Takeda, BiolineRx, Janssen Other Relationship: CellerantRavi VijConsulting or Advisory Role: Bristol Myers Squibb, Celgene, Janssen, Sanofi, Karyopharm Therapeutics, Takeda, Genentech, AbbVie, Oncopeptides Research Funding: Takeda, Celgene, Bristol Myers Squibb Travel, Accommodations, Expenses: Celgene, Bristol Myers Squibb, Sanofi, Janssen, DAVAOncology, Karyopharm Therapeutics, Amgen, Takeda, AbbVieAmanda F. CashenConsulting or Advisory Role: Agios Speakers' Bureau: Novartis, CelgeneIskra PusicConsulting or Advisory Role: Kadmon, Incyte, SyndaxCamille N. AbboudStock and Other Ownership Interests: AbbVie (I), Abbott Laboratories (I), Gilead Sciences (I), Bristol Myers Squibb (I), Johnson & Johnson (I) Honoraria: Cardinal Health, Dova Pharmaceuticals, NkartaTherapeutics, Archer Biosciences Research Funding: Actinium Pharmaceuticals, Selvita (Inst), AlloVir (Inst), Forty-Seven (Inst)Armin GhobadiHonoraria: Kite Pharma Consulting or Advisory Role: Kite Pharma, Celgene, Amgen, Wugen Speakers' Bureau: Kite Pharma, AmgenGeoffrey L. UyConsulting or Advisory Role: Astellas Pharma, Genentech, Jazz PharmaceuticalsMark A. SchroederConsulting or Advisory Role: Astellas Pharma, Dova Pharmaceuticals, Flatiron Health, GlaxoSmithKline, Incyte, Partners Therapeutics, Partners Therapeutics, Pfizer Speakers' Bureau: AbbVie, Merck, TakedaJohn F. DiPersioStock and Other Ownership Interests: Magenta Therapeutics, Wugen Honoraria: Incyte Consulting or Advisory Role: Cellworks, Rivervest, Magenta Therapeutics, Incyte Research Funding: Amphivena Therapeutics (Inst), Macrogenics (Inst), Incyte (Inst), Wugen (Inst), BiolineRx (Inst), Maxcyte (Inst), Bigelow Aerospace (Inst) Patents, Royalties, Other Intellectual Property: Patents: CD7 and CD2 knockout for CART to CD7 and CDL; Duvelisib for treatment of cytokine release syndrome; NT-17 to enhance CART survival; Novel WU mobilizing compounds (Inst); Selection of IMPDH mutant stem cells; IFNγ, upregulate MHCII for relapsed AML; Dextran-based molecules to detect CAR-T cells; Combining integrin inhibitor with chemokine binders, 016131; JAK and calcineurin inhibition, solid organ transplant; VLA4, gro-b; Triple combination–CXCR2, VLA-4, gro-b; Targeting IFNR/CSCR3 in GvHD; WU/SLU compounds VLA4 and CXCR2 (Inst) Travel, Accommodations, Expenses: Incyte, Macrogenics, Magenta TherapeuticsMary C. PolitiResearch Funding: MerckDavid H. SpencerConsulting or Advisory Role: Wugen Research Funding: IlluminaEric J. DuncavageStock and Other Ownership Interests: P&V Licensing Consulting or Advisory Role: Cofactor Genomics, Bristol Myers Squibb, Eli Lilly Open Payments Link: https://openpaymentsdata.cms.gov/physician/317379Timothy J. LeyResearch Funding: Rigel Patents, Royalties, Other Intellectual Property: Licenses for monoclonal antibodies and recombinant granzymesMeagan A. JacobyHonoraria: Takeda Research Funding: Geron (Inst), AbbVie (Inst), Jazz Pharmaceuticals (Inst), Aprea AB (Inst), Amphivena (Inst), Janssen (Inst) No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Natural killer (NK) cells are an emerging cancer cellular therapy and potent mediators of antitumor immunity. Cytokine-induced memory-like (ML) NK cellular therapy is safe and induces remissions in patients with acute myeloid leukemia (AML). However, the dynamic changes in phenotype that occur after NK-cell transfer that affect patient outcomes remain unclear. Here, we report comprehensive multidimensional correlates from ML NK cell-treated patients with AML using mass cytometry. These data identify a unique in vivo differentiated ML NK-cell phenotype distinct from conventional NK cells. Moreover, the inhibitory receptor NKG2A is a dominant, transcriptionally induced checkpoint important for ML, but not conventional NK-cell responses to cancer. The frequency of CD8α + donor NK cells is negatively associated with AML patient outcomes after ML NK therapy. Thus, elucidating the multidimensional dynamics of donor ML NK cells in vivo revealed critical factors important for clinical response, and new avenues to enhance NK-cell therapeutics. SIGNIFICANCE: Mass cytometry reveals an in vivo memory-like NK-cell phenotype, where NKG2A is a dominant checkpoint, and CD8α is associated with treatment failure after ML NK-cell therapy. These findings identify multiple avenues for optimizing ML NK-cell immunotherapy for cancer and define mechanisms important for ML NK-cell function. This article is highlighted in the In This Issue feature, p. 1775 ., (©2020 American Association for Cancer Research.)

Decitabine has been explored as a reduced-intensity therapy for older or unfit patients with acute myeloid leukemia (AML). To better understand the risk of infections during decitabine treatment, we retrospectively examined the culture results from each infection-related serious adverse event that occurred among 85 AML and myelodysplastic syndromes (MDS) patients treated in a prospective clinical study using 10-day cycles of decitabine at Washington University School of Medicine. Culture results were available for 163 infection-related complications that occurred in 70 patients: 90 (55.2%) events were culture-negative, 32 (19.6%) were gram-positive bacteria, 20 (12.3%) were gram-negative bacteria, 12 (7.4%) were mixed, 6 (3.7%) were viral, 2 (1.2%) were fungal, and 1 (0.6%) was mycobacterial. Infection-related mortality occurred in 3/24 (13%) of gram-negative events, and 0/51 gram-positive events. On average, nearly one third of patients experienced an infection-related complication with each cycle, and the incidence did not decrease during later cycles. In summary, in patients receiving 10-day decitabine, infectious complications are common and may occur during any cycle of therapy. Although febrile events are commonly culture-negative, gram-positive infections are the most frequent source of culture-positive infections, but gram-negative infections represent a significant risk of mortality in AML and MDS patients treated with decitabine., (© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: The molecular determinants of clinical responses to decitabine therapy in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) are unclear., Methods: We enrolled 84 adult patients with AML or MDS in a single-institution trial of decitabine to identify somatic mutations and their relationships to clinical responses. Decitabine was administered at a dose of 20 mg per square meter of body-surface area per day for 10 consecutive days in monthly cycles. We performed enhanced exome or gene-panel sequencing in 67 of these patients and serial sequencing at multiple time points to evaluate patterns of mutation clearance in 54 patients. An extension cohort included 32 additional patients who received decitabine in different protocols., Results: Of the 116 patients, 53 (46%) had bone marrow blast clearance (<5% blasts). Response rates were higher among patients with an unfavorable-risk cytogenetic profile than among patients with an intermediate-risk or favorable-risk cytogenetic profile (29 of 43 patients [67%] vs. 24 of 71 patients [34%], P<0.001) and among patients with TP53 mutations than among patients with wild-type TP53 (21 of 21 [100%] vs. 32 of 78 [41%], P<0.001). Previous studies have consistently shown that patients with an unfavorable-risk cytogenetic profile and TP53 mutations who receive conventional chemotherapy have poor outcomes. However, in this study of 10-day courses of decitabine, neither of these risk factors was associated with a lower rate of overall survival than the rate of survival among study patients with intermediate-risk cytogenetic profiles., Conclusions: Patients with AML and MDS who had cytogenetic abnormalities associated with unfavorable risk, TP53 mutations, or both had favorable clinical responses and robust (but incomplete) mutation clearance after receiving serial 10-day courses of decitabine. Although these responses were not durable, they resulted in rates of overall survival that were similar to those among patients with AML who had an intermediate-risk cytogenetic profile and who also received serial 10-day courses of decitabine. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT01687400 .).

Clofarabine has shown activity and tolerability in older patients with acute myeloid leukemia (AML). We investigated the safety and tolerability of an oral formulation of clofarabine for consolidation therapy of patients aged 60 and older with AML. In this phase I study, twenty-two patients older than 60 years with AML in first complete remission were treated once daily with oral clofarabine for 14 or 21 days of a 28-day cycle, for up to five cycles. Dose escalation from 1 mg to 6 mg daily using a 3 + 3 design was used to determine dose-limiting toxicities (DLT), the maximum tolerated dose (MTD), and tolerability of oral clofarabine. No DLTs or Grade 3-4 nonhematologic toxicities were observed. The primary toxicities were hematologic, including uncomplicated grade 3-4 neutropenia (50%) and thrombocytopenia (50%). Given that myelosuppression necessitating dose delays/reductions was observed more commonly at higher doses, the recommended phase II dose is 2 mg daily for 21 of 28 days. At doses equal to or greater than 2 mg, the median relapse-free survival was 28.35 months. Oral clofarabine was well-tolerated with encouraging activity in patients older than 60 years. Further investigation of oral clofarabine as a consolidation and/or maintenance therapy in AML for older individuals is warranted. (ClinicalTrials.gov:NCT00727766)., (Copyright © 2014 Wiley Periodicals, Inc.)

Altered microRNA (miRNA) expression is frequently observed in acute myelogenous leukemia (AML) and has been implicated in leukemic transformation. Whether somatic copy number alterations (CNAs) are a frequent cause of altered miRNA gene expression is largely unknown. Herein, we used comparative genomic hybridization with a custom high-resolution miRNA-centric array and/or whole-genome sequence data to identify somatic CNAs involving miRNA genes in 113 cases of AML, including 50 cases of de novo AML, 18 cases of relapsed AML, 15 cases of secondary AML following myelodysplastic syndrome, and 30 cases of therapy-related AML. We identified a total of 48 somatic miRNA gene-containing CNAs that were not identified by routine cytogenetics in 20 patients (18%). All these CNAs also included one or more protein coding genes. We identified a single case with a hemizygous deletion of MIR223, resulting in the complete loss of miR-223 expression. Three other cases of AML were identified with very low to absent miR-223 expression, an miRNA gene known to play a key role in myelopoiesis. However, in these cases, no somatic genetic alteration of MIR223 was identified, suggesting epigenetic silencing. These data show that somatic CNAs specifically targeting miRNA genes are uncommon in AML.

Chromosomal deletions and amplifications that occur in affected cells from patients with myelodysplastic syndromes and acute myeloid leukemia often contain genes that contribute to disease pathogenesis. Identification of copy number alterations (deletions and amplifications) and regions of copy neutral loss of heterozygosity using array-based platforms has led to the identification of genes that are commonly mutated in myeloid malignancies. In this article, we review the literature and highlight the array-based studies that directly compare matched normal and tumor samples from the same individual to identify somatic alterations. We also discuss the use of next-generation sequencing to identify all types of structural variants, including copy number alterations and copy neutral loss of heterozygosity, and provide an outlook for how this technology may be used to interrogate cancer genomes.