Acute megakaryoblastic leukaemia shows high frequency of chromosome 1q aberrations and dismal outcome.

Acute megakaryoblastic leukaemia (AMKL) is associated with poor prognosis. Limited information is available on its cytogenetics, molecular genetics and clinical outcome. We performed genetic analyses, evaluated prognostic factors and the value of allogeneic haematopoietic stem cell transplantation (allo-HSCT) in a homogenous adult AMKL patient cohort. We retrospectively analysed 38 adult patients with AMKL (median age: 58 years, range: 21-80). Most received intensive treatment in AML Cooperative Group (AMLCG) trials between 2001 and 2016. Cytogenetic data showed an accumulation of adverse risk markers according to ELN 2017 and an unexpected high frequency of structural aberrations on chromosome arm 1q (33%). Most frequently, mutations occurred in TET2 (23%), TP53 (23%), JAK2 (19%), PTPN11 (19%) and RUNX1 (15%). Complete remission rate in 33 patients receiving intensive chemotherapy was 33% and median overall survival (OS) was 33 weeks (95% CI: 21-45). Patients undergoing allo-HSCT (n = 14) had a superior median OS (68 weeks; 95% CI: 11-126) and relapse-free survival (RFS) of 27 weeks (95% CI: 4-50), although cumulative incidence of relapse after allo-HSCT was high (62%). The prognosis of AMKL is determined by adverse genetic risk factors and therapy resistance. So far allo-HSCT is the only potentially curative treatment option in this dismal AML subgroup.

HSP90 inhibition overcomes ibrutinib resistance in mantle cell lymphoma.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib induces responses in 70% of patients with relapsed and refractory mantle cell lymphoma (MCL). Intrinsic resistance can occur through activation of the nonclassical NF-κB pathway and acquired resistance may involve the BTK C481S mutation. Outcomes after ibrutinib failure are dismal, indicating an unmet medical need. We reasoned that newer heat shock protein 90 (HSP90) inhibitors could overcome ibrutinib resistance by targeting multiple oncogenic pathways in MCL. HSP90 inhibition induced the complete degradation of both BTK and IκB kinase α in MCL lines and CD40-dependent B cells, with downstream loss of MAPK and nonclassical NF-κB signaling. A proteome-wide analysis in MCL lines and an MCL patient-derived xenograft identified a restricted set of targets from HSP90 inhibition that were enriched for factors involved in B-cell receptor and JAK/STAT signaling, the nonclassical NF-κB pathway, cell-cycle regulation, and DNA repair. Finally, multiple HSP90 inhibitors potently killed MCL lines in vitro, and the clinical agent AUY922 was active in vivo against both patient-derived and cell-line xenografts. Together, these findings define the HSP90-dependent proteome in MCL. Considering the disappointing clinical activity of HSP90 inhibitors in other contexts, trials in patients with MCL will be essential for defining the efficacy of and mechanisms of resistance after ibrutinib failure.

Modeling early treatment response in AML from cell-free tumor DNA.

Monitoring disease response after intensive chemotherapy for acute myeloid leukemia (AML) currently requires invasive bone marrow biopsies, imposing a significant burden on patients. In contrast, cell-free tumor DNA (ctDNA) in peripheral blood, carrying tumor-specific mutations, offers a less-invasive assessment of residual disease. However, the relationship between ctDNA levels and bone marrow blast kinetics remains unclear. We explored this in 10 AML patients with NPM1 and IDH2 mutations undergoing initial chemotherapy. Comparison of mathematical mixed-effect models showed that (1) inclusion of blast cell death in the bone marrow, (2) transition of ctDNA to peripheral blood, and (3) ctDNA decay in peripheral blood describes kinetics of blast cells and ctDNA best. The fitted model allows prediction of residual bone marrow blast content from ctDNA, and its scaling factor, representing clonal heterogeneity, correlates with relapse risk. Our study provides precise insights into blast and ctDNA kinetics, offering novel avenues for AML disease monitoring.

Double Drop-Off Droplet Digital PCR: A Novel, Versatile Tool for Mutation Screening and Residual Disease Monitoring in Acute Myeloid Leukemia Using Cellular or Cell-Free DNA.

In acute myeloid leukemia (AML), somatic gene mutations are important prognostic markers and increasingly constitute therapeutic targets. Therefore, robust, sensitive, and fast diagnostic assays are needed. Current techniques for mutation screening and quantification, including next-generation sequencing and quantitative PCR, each have weaknesses that leave a need for novel diagnostic tools. We established double drop-off digital droplet PCR (DDO-ddPCR) assays for gene mutations in NPM1, IDH2, and NRAS, which can detect and quantify diverse alterations at two nearby hotspot regions present in these genes. These assays can be used for mutation screening as well as quantification and sequential monitoring. The assays were validated against next-generation sequencing and existing ddPCR assays and achieved high concordance with an overall sensitivity comparable to conventional digital PCR. In addition, the feasibility of detecting and monitoring genetic alterations in peripheral blood cell-free DNA (cfDNA) of patients with AML by DDO-ddPCR was studied. cfDNA analysis was found to have similar sensitivity compared to quantitative PCR-based analysis of peripheral blood. Finally, the cfDNA-based digital PCR in several clinical scenarios was found to be useful in long-term monitoring of target-specific therapy, early response assessment during induction chemotherapy, and identification of mutations in patients with extramedullary disease. Thus, DDO-ddPCR-based cfDNA analysis may complement existing genetic tools for diagnosis and disease monitoring in AML.

The clinical mutatome of core binding factor leukemia.

The fusion genes CBFB/MYH11 and RUNX1/RUNX1T1 block differentiation through disruption of the core binding factor (CBF) complex and are found in 10-15% of adult de novo acute myeloid leukemia (AML) cases. This AML subtype is associated with a favorable prognosis; however, nearly half of CBF-rearranged patients cannot be cured with chemotherapy. This divergent outcome might be due to additional mutations, whose spectrum and prognostic relevance remains hardly defined. Here, we identify nonsilent mutations, which may collaborate with CBF-rearrangements during leukemogenesis by targeted sequencing of 129 genes in 292 adult CBF leukemia patients, and thus provide a comprehensive overview of the mutational spectrum ('mutatome') in CBF leukemia. Thereby, we detected fundamental differences between CBFB/MYH11- and RUNX1/RUNX1T1-rearranged patients with ASXL2, JAK2, JAK3, RAD21, TET2, and ZBTB7A being strongly correlated with the latter subgroup. We found prognostic relevance of mutations in genes previously known to be AML-associated such as KIT, SMC1A, and DHX15 and identified novel, recurrent mutations in NFE2 (3%), MN1 (4%), HERC1 (3%), and ZFHX4 (5%). Furthermore, age >60 years, nonprimary AML and loss of the Y-chromosomes are important predictors of survival. These findings are important for refinement of treatment stratification and development of targeted therapy approaches in CBF leukemia.

Genetic heterogeneity of cytogenetically normal AML with mutations of CEBPA.

Biallelic mutations of the CCAAT/enhancer binding protein α (CEBPA) gene define a distinct genetic entity of acute myeloid leukemia (AML) with favorable prognosis. The presence of GATA2 and CSF3R mutations that are specifically associated with this subgroup but not mutated in all samples suggests a genetic heterogeneity of biCEBPA-mutated AML. We characterized the mutational landscape of CEBPA-mutated cytogenetically normal AML by targeted amplicon resequencing. We analyzed 48 biallelically mutated CEBPA (biCEBPA), 32 monoallelically mutated CEBPA (moCEBPA), and 287 wild-type CEBPA (wtCEBPA) patient samples from German AML Cooperative Group studies or registry. Targeted sequencing of 42 genes revealed that moCEBPA patients had significantly more additional mutations and additional mutated genes than biCEBPA patients. Within the group of biCEBPA patients, we identified 2 genetic subgroups defined by the presence or absence of mutations in chromatin/DNA modifiers (C), cohesin complex (C), and splicing (S) genes: biCEBPA CCSpos (25/48 [52%]) and biCEBPA CCSneg (23/48 [48%]). Equivalent subgroups were identified in 51 biCEBPA patients from the Cancer Genome Project. Patients in the biCEBPA CCSpos group were significantly older and had poorer overall survival and lower complete remission rates following intensive chemotherapy regimens compared with patients in the biCEBPA CCSneg group. Patients with available remission samples from the biCEBPA CCSpos group cleared the biCEBPA mutations, but most had persisting CCS mutations in complete remission, suggesting the presence of a preleukemic clone. In conclusion, CCS mutations define a distinct biological subgroup of biCEBPA AML that might refine prognostic classification of AML. This trial was registered at www.clinicaltrials.gov as #NCT00266136 and NCT01382147.

Acetylation of C/EBPα inhibits its granulopoietic function.

CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBPα at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBPα DNA-binding ability and modulates C/EBPα transcriptional activity. Acetylated C/EBPα is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBPα mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBPα-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBPα and demonstrate the importance of C/EBPα acetylation in myeloid differentiation.