Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has ...suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later
. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.
Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme ...isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617F and mutant IDH1R132H or Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617F Idh2R140Q-mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mut and IDH2mut mutations. Taken together, these data suggest that combined JAK and IDH inhibition may offer a therapeutic advantage in this high-risk MPN subtype.
Measurable residual disease is associated with inferior outcomes in patients with acute myeloid leukemia (AML). Measurable residual disease monitoring enhances risk stratification and may guide ...therapeutic intervention. The European LeukemiaNet working party recently came to a consensus recommendation incorporating leukemia associated immunophenotype-based different from normal approach by multi-color flow cytometry for measurable residual disease evaluation. However, the analytical approach is highly expertise-dependent and difficult to standardize. Here we demonstrate that loss of plasmacytoid dendritic cell differentiation after 7+3 induction in AML is highly specific for measurable residual disease positivity (specificity 97.4%) in a uniformly treated patient cohort. Moreover, loss of plasmacytoid dendritic cell differentiation as determined by a blast-to-plasmacytoid dendritic cell ratio >10 was strongly associated with inferior overall and relapse-free survival (RFS) Hazard ratio 2.79, 95% confidence interval (95%CI): 0.98-7.97;
=0.077) and 3.83 (95%CI: 1.51-9.74;
=0.007), respectively), which is similar in magnitude to measurable residual disease positivity. Importantly, measurable residual disease positive patients who reconstituted plasmacytoid dendritic cell differentiation (blast/ plasmacytoid dendritic cell ratio <10) showed a higher rate of measurable residual disease clearance at later pre-transplant time points compared to patients with loss of plasmacytoid dendritic cell differentiation (blast/ plasmacytoid dendritic cell ratio <10) (6 of 12, 50%
2 of 18, 11%;
=0.03). Furthermore pre-transplant plasmacytoid dendritic cell recovery was associated with superior outcome in measurable residual disease positive patients. Our study provides a novel, simple, broadly applicable, and quantitative multi-color flow cytometry approach to risk stratification in AML.
Therapy-related myelodysplastic syndrome and acute leukemias (t-MDS/AL) are a major cause of nonrelapse mortality among pediatric cancer survivors. Although the presence of clonal hematopoiesis (CH) ...in adult patients at cancer diagnosis has been implicated in t-MDS/AL, there is limited published literature describing t-MDS/AL development in children.
We performed molecular characterization of 199 serial bone marrow samples from 52 patients treated for high-risk neuroblastoma, including 17 with t-MDS/AL (transformation), 14 with transient cytogenetic abnormalities (transient), and 21 without t-MDS/AL or cytogenetic alterations (neuroblastoma-treated control). We also evaluated for CH in a cohort of 657 pediatric patients with solid tumor.
We detected at least one disease-defining alteration in all cases at t-MDS/AL diagnosis, most commonly TP53 mutations and KMT2A rearrangements, including involving two novel partner genes (PRDM10 and DDX6). Backtracking studies identified at least one t-MDS/AL-associated mutation in 13 of 17 patients at a median of 15 months before t-MDS/AL diagnosis (range, 1.3-32.4). In comparison, acquired mutations were infrequent in the transient and control groups (4/14 and 1/21, respectively). The relative risk for development of t-MDS/AL in the presence of an oncogenic mutation was 8.8 for transformation patients compared with transient. Unlike CH in adult oncology patients, TP53 mutations were only detectable after initiation of cancer therapy. Last, only 1% of pediatric patients with solid tumor evaluated had CH involving myeloid genes.
These findings demonstrate the clinical relevance of identifying molecular abnormalities in predicting development of t-MDS/AL and should guide the formation of intervention protocols to prevent this complication in high-risk pediatric patients.
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− remission, while mutations in TP53, SF3B1, ASXL1, and RUNX1 and karyotypic abnormalities including inv (3), monosomy 5 or 7 predicted low rates of MRD− remission. 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.
Covalent BTK inhibitors (BTKi) have transformed the management of mantle cell lymphoma (MCL), but most patients will require additional treatment. Pirtobrutinib is a highly selective, non-covalent ...(reversible) BTKi that inhibits both wild-type and C481-mutated BTK with equal low nM potency.
To evaluate pirtobrutinib safety and efficacy in patients with MCL.
BRUIN is an ongoing multicenter phase 1/2 study (NCT03740529) of pirtobrutinib monotherapy.
Global; community hospitals, academic medical centers.
Patients with advanced B-cell malignancies.
Oral pirtobrutinib, phase 1 dose-escalated in a standard 3+3 design, phase 2 continuous therapy, 28-day cycles.
The primary phase 1 objective was to determine the recommended phase 2 dose (RP2D) and the primary phase 2 objective was overall response rate (ORR); secondary objectives included duration of response, progression-free survival, overall survival, safety/tolerability, and pharmacokinetics.
As of 27 September 2020, 323 patients (170 CLL/SLL, 61 MCL, 26 WM, 26 DLBCL, 13 MZL, 12 FL, 9 RT, and 6 other NHL) were treated on 7 dose levels (25-300mg QD). No DLTs were reported and MTD was not reached (n=323). 200mg QD was selected as the RP2D. Fatigue (20%), diarrhea (17%) and contusion (13%) were the most frequent treatment-emergent adverse events regardless of attribution or grade seen in >10% of patients. The most common adverse event of grade ≥3 was neutropenia (10%). Five (1%) patients discontinued due to treatment-related adverse events. 52 prior BTKi treated MCL patients were efficacy evaluable with an ORR of 52% (95% CI 38-66; 13 CR 25%, 14 PR 27%, 9 SD 17%), 11 PD 21% and 5 10% discontinued prior to first response assessment). Median follow-up was 6 months (0.7-18.3+). Responses were observed in 9/14 patients (64%) with prior autologous or allogeneic stem cell transplant, and 2/2 with prior CAR-T cell therapy.
Pirtobrutinib demonstrated promising efficacy in heavily pretreated, poor-prognosis MCL following multiple prior lines of therapy, including a covalent BTKi. Pirtobrutinib was well tolerated and exhibited a wide therapeutic index. Updated data, including approximately 60 new patients with MCL and an additional 10 months since the prior data-cut will be presented.
The genetic aberrations that drive mixed phenotype acute leukemia (MPAL) remain largely unknown, with the exception of a small subset of MPALs harboring BCR-ABL1 and MLL translocations. We performed ...clinicopathologic and genetic evaluation of 52 presumptive MPAL cases at Memorial Sloan Kettering Cancer Center. Only 29 out of 52 (56%) cases were confirmed to be bona fide MPAL according to the 2016 World Heath Organization classification. We identified PHF6 and DNMT3A mutations as the most common recurrent mutations in MPAL, each occurring in 6 out of 26 (23%) cases. These mutations are mutually exclusive of each other and BCR-ABL1/MLL translocations. PHF6- and DNMT3A-mutated MPAL showed marked predilection for T-lineage differentiation (5/6 PHF6 mutated, 6/6 DNMT3A mutated). PHF6-mutated MPAL occurred in a younger patient cohort compared with DNMT3A-mutated cases (median age, 27 years vs 61 years, P < .01). All 3 MPAL cases with both T- and B-lineage differentiation harbored PHF6 mutations. MPAL with T-lineage differentiation was associated with nodal or extramedullary involvement (9/15 60% vs 0, P = .001) and a higher relapse incidence (78% vs 22%, P = .017) compared with those without T-lineage differentiation. Sequencing studies on flow-cytometry–sorted populations demonstrated that PHF6 mutations are present in all blast compartments regardless of lineage differentiation with high variant allele frequency, implicating PHF6 as an early mutation in MPAL pathogenesis. In conclusion, PHF6 and DNMT3A mutations are the most common somatic alterations identified in MPAL and appear to define 2 distinct subgroups of MPAL with T-lineage differentiation with inferior outcomes.
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Stably acquired mutations in hematopoietic cells represent substrates of selection that may lead to clonal hematopoiesis (CH), a common state in cancer patients that is associated with a heightened ...risk of leukemia development. Owing to technical and sample size limitations, most CH studies have characterized gene mutations or mosaic chromosomal alterations (mCAs) individually. Here we leverage peripheral blood sequencing data from 32,442 cancer patients to jointly characterize gene mutations (n = 14,789) and mCAs (n = 383) in CH. Recurrent composite genotypes resembling known genetic interactions in leukemia genomes underlie 23% of all detected autosomal alterations, indicating that these selection mechanisms are operative early in clonal evolution. CH with composite genotypes defines a patient group at high risk of leukemia progression (3-year cumulative incidence 14.6%, CI: 7-22%). Multivariable analysis identifies mCA as an independent risk factor for leukemia development (HR = 14, 95% CI: 6-33, P < 0.001). Our results suggest that mCA should be considered in conjunction with gene mutations in the surveillance of patients at risk of hematologic neoplasms.
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