Marcucci, Guido

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal expansion of myeloid blasts in the peripheral blood, bone marrow, and/or other tissues. It is the most common form of acute leukemia among adults and accounts for the largest number of annual deaths from leukemias in the United States. 1 The NCCN Guidelines Panel for AML convenes annually to update recommendations for the diagnosis and treatment of AML in adults. These recommendations are based on a review of recently published clinical trials that have led to significant improvements in treatment or have yielded new information regarding biologic factors that may have prognostic importance. These NCCN Guidelines Insights summarize the panel’s most recent recommendations regarding the monitoring and management of measurable (minimal) residual disease.

Abstract PAX5 is genetically altered in over 30% of B-ALL cases, with point mutations representing the second most common type of alteration. These point mutations are highly enriched in the DNA binding (paired) domain and have been recognized as founder events in PAX5 P80R and PAX5alt B-ALL subtypes, which account for over 12% of B-ALL. However, the molecular features and underlying mechanisms of these mutations in B-ALL remain largely undefined, limiting the development of targeted therapies and resulting in poor outcomes, especially in adult patients. We systematically investigated the functional roles and oncogenic mechanisms of PAX5 paired domain mutations. Integrated CRISPR screening and genomic profiling revealed that the paired domain is critical for PAX5 function. Disruptions in this region significantly reduced cell viability in B-ALL cell lines, and analysis of 2,955 B-ALL samples showed that recurrent PAX5 missense mutations are highly enriched in this domain, further underscoring its functional importance. Functional analysis of knock-in mice with PAX5 mutations revealed that R38H and R140L severely impair DNA binding and block B-cell differentiation, while P34L and P80R retain binding ability but alter binding specificity and gene regulation. Mice carrying homozygous (P34L, R38H, P80R, R140L) or heterozygous (R38H, P80R) mutations developed spontaneous B-ALL within one year. To model co-occurring mutations observed in patients, Pax5 R38H/P34L and R38H/R140L mice were generated. These compound mutations exhibited greater leukemogenic potential than single mutants. Transcriptomic analysis of leukemia samples showed consistent up-regulation of metabolic pathways and Myc target genes. Notably, biallelic PAX5 alterations and additional genetic lesions in signaling pathways were detected in all PAX5R38H/+ and PAX5P80R/+ leukemias, faithfully recapitulating the genetic alteration features of patient B-ALL driven by PAX5 mutations. Among Pax5-mutant mouse strains, Pax5P80R/+ mice develop B-ALL with short latency and complete penetrance, making them an ideal model to study the mutational evolution and leukemogenesis of B-ALL. To characterize the stepwise leukemogenesis process, we performed serial bone marrow aspirations in the same Pax5P80R/+ mice. In all 17 mice analyzed, we identified early preleukemic clones with only Pax5 WT deletions, followed by the emergence of leukemic clones harboring Jak1/3 mutations. Single-cell RNA/BCR-seq revealed that Pax5 WT allele deletions relieve metabolic constraints, thereby promoting clonal expansion of preleukemic B cells blocked at the pre-B stage. These changes coincide with downregulation of metabolic checkpoint genes and upregulation of glycolytic enzymes, promoting genetic instability and facilitating malignant transformation. Clinically, PAX5 P80R patients have better outcomes than PAX5alt cases. Using our mouse models, we observed that leukemic cells driven by the PAX5 P80R mutation exhibit greater sensitivity to dexamethasone compared to the ones carrying other PAX5 mutations in PAX5alt subtype, potentially due to their heightened dependence on glucose uptake and glycolysis, suggesting that metabolic rewiring driven by PAX5 mutants may influence therapeutic response. Interestingly, we found that an aberrant isoform of MEGF10 (aMEGF10) is highly and specifically expressed in PAX5 P80R B-ALL. This N-terminal truncated isoform encodes a protein that retains the intracellular immunoreceptor tyrosine-based activation motifs (ITAMs). Using CUT&Tag, we showed that PAX5 P80R directly binds to the aMEGF10 promoter. By engineering human CD34⁺ cells, we established a humanized PAX5 P80R leukemia model that recapitulates the gene expression profile of patient samples, including robust activation of aMEGF10. Functionally, aMEGF10 binds to and activates SYK, thereby promoting leukemic cell proliferation. Importantly, this oncogenic effect depends on intact ITAM motifs and SYK signaling, revealing a novel targetable vulnerability in PAX5 P80R leukemias. Overall, the study establishes that PAX5 DNA binding domain mutations contribute to leukemogenesis through stepwise loss of PAX5 WT alleles and acquisition of signaling pathway mutations, involving impaired differentiation, metabolic deregulation, and novel oncogene activation. These findings not only deepen the understanding of PAX5-driven leukemias but also highlight aMEGF10–SYK signaling as a promising therapeutic target.

Abstract BMT CTN (Blood and Marrow Transplant Clinical Trials Network) 1506 (“MORPHO”) was a randomized study of gilteritinib compared with placebo as maintenance therapy after hematopoietic cell transplantation (HCT) for patients with FLT3-ITD–mutated acute myeloid leukemia (AML). A key secondary end point was to determine the impact on survival of before and/or after HCT measurable residual disease (MRD), as determined using a highly sensitive assay for FLT3-ITD mutations. Generally, gilteritinib maintenance therapy was associated with improved relapse-free survival (RFS) for participants with detectable peri-HCT MRD, whereas no benefit was evident for those lacking detectable MRD. We conducted a post hoc analysis of the data and found that the level of MRD detected with this approach correlated remarkably with RFS and relapse risk, and that MRD detectable at any level negatively affected RFS. In the placebo arm, 42.2% of participants with detectable FLT3-ITD MRD relapsed compared with 13.4% of those without detectable MRD. We found that 14.8% of participants had multiple FLT3-ITD clones detected as MRD and had worse survival irrespective of treatment arm. Finally, we examined the kinetics of FLT3-ITD clonal relapse or eradication and found that participants on the placebo arm with detectable MRD relapsed rapidly after HCT, often within a few weeks. MRD-positive participants on the gilteritinib arm relapsed either with FLT3 wild-type clones (assessed by capillary electrophoresis), after cessation of gilteritinib with persistent MRD, or on progression of multiclonal disease. These data demonstrate the potential of FLT3-ITD MRD to guide therapy with gilteritinib for this subtype of AML. This trial was registered at www.clinicaltrials.gov as #NCT02997202.

Background: C10603/RATFIY was the first AML trial to show the benefit of adding a targeted agent to intensive chemotherapy for a specific genetically determined subset (Stone R et al, NEJM 2017). The addition of the multi-kinase inhibitor midostaurin (M) to chemotherapy in previously untreated adults with FLT3-mutant AML resulted in superior event-free (EFS) and overall survival (OS) compared to placebo (P) and led to the approval of this agent in combination therapy. Subsequently, additional targeted drugs including gilteritinib for relapsed/refractory FLT3-mutant AML and quizartinib plus chemotherapy in untreated adults with AML with FLT3-ITD disease have gained approval. Long-term follow-up after reporting of the primary endpoint, especially if met, is important in oncology. Objective: We report the EFS and OS data from C10603 using 10 years of follow up to determine the persistence of a midostaurin benefit and to assess the nature of late relapses or toxicity. Methods: C10603 enrolled 717 pts (360 on the M and 357 on the P arm; median age 47.8 years (range 18-61), 398 women (55.5%) of whom 51.7% were randomized to M and 59.4% to P (p=0.04), and 89% white) from 2011-2015 with previously untreated AML who had either a FLT3-TKD or ITD mutation with allelic ratio of >0.05 to receive daunorubicin/cytarabine (3+7) induction (one reinduction permitted) followed by up to 4 consolidation cycles with cytarabine 3 g/m2 every 12h on days 1, 3 and 5. M (50 mg bid) or P was given orally on days 8-22 of each chemotherapy cycle as well as daily during one year of single-agent maintenance. Allogeneic transplantation (alloHCT) was employed at investigator's discretion; study drug was not continued post-HCT. Pts stayed on their randomized arm and were not re-randomized prior to maintenance. Pts were followed for relapse and survival after the end of protocol therapy, regardless of subsequent therapy, for 10 years. Log-rank tests, Kaplan-Meier methods, and Cox proportional hazard models were used to evaluate the treatment effect in EFS and OS, as well as the alloHCT effect. All p values are two-sided. Results: We have completed the 10-year FU on this trial and data collection has concluded. The median EFS was 8.2 months (95% CI, 5.5-11.4) on the M arm compared with 3.0 months for P (95% CI, 1.9-6.0) with a HR of 0.79 (95% CI 0.67-0.94, p=0.0067). Among the 420 CR pts who achieved complete remission (CR) by the protocol specified time of 60 days, 253 have had an event (death in 69 and relapse in 184); these events were relatively equal on each arm. OS, the co-primary endpoint, remained marginally superior for those randomized to M compared to P; the 10-year OS estimate was 43.7% (95% CI, 38.7-49.3%) vs 38.6% (95% CI, 33.6-44.4%) (p= 0.0485). The hazard ratios for OS favored randomization to M in each key biological subgroup (FLT3-ITD low or high allelic ratio or FLT3-TKD), but smaller subsets precluded statistical significance. There was a 10-year OS benefit in favor of M in men (p=0.005), but not women (p=0.9). A total of 414 transplants were performed: only 5 in the latter half of the follow-up period. Transplantation in CR1 was highly beneficial overall (10-year OS, 56% vs 35.8% without transplantation, p=0.001). Among those transplanted in CR1, randomization to M was marginally better than P (61.5% vs 49%, p=.064). After censoring for alloHCT in CR1, there was an OS trend in favor of M (51.8% v 48.0%, p=0.102). In CR1 patients who were randomized to maintenance therapy, M reduced the cumulative incidence of relapse in ELN 2017 favorable and intermediate risk pts, but not in those with adverse risk disease (HR 0.71, 0.47, and 1.01, respectively); maintenance M had no effect on OS. Only 3 pts relapsed after 5 years (all randomized to M). 25 pts randomized to M vs 13 P pts died after 5 years of follow-up. 5 pts on each arm died of a new primary cancer whereas 17 M and 5 P pts were coded as having died from treatment-related causes. None of the 3 pts who relapsed after 4 years had spliceosome mutations at diagnosis; whereas 18% (21/117) patients pts who had relapsed earlier had spliceosome mutations at diagnosis (p=0.419). Conclusions: The EFS benefit of randomization to midostaurin vs placebo when added to chemotherapy was maintained over time, although the benefit for OS was diminished, likely due in part to aging. Patient and disease factors differed between early vs late relapses, which could suggest the use of alternative therapies during distinct therapeutic stages.

PURPOSE Allogeneic hematopoietic cell transplantation (HCT) improves outcomes for patients with AML harboring an internal tandem duplication mutation of FLT3 ( FLT3-ITD) AML. These patients are routinely treated with a FLT3 inhibitor after HCT, but there is limited evidence to support this. Accordingly, we conducted a randomized trial of post-HCT maintenance with the FLT3 inhibitor gilteritinib (ClinicalTrials.gov identifier: NCT02997202 ) to determine if all such patients benefit or if detection of measurable residual disease (MRD) could identify those who might benefit. METHODS Adults with FLT3-ITD AML in first remission underwent HCT and were randomly assigned to placebo or 120 mg once daily gilteritinib for 24 months after HCT. The primary end point was relapse-free survival (RFS). Secondary end points included overall survival (OS) and the effect of MRD pre- and post-HCT on RFS and OS. RESULTS Three hundred fifty-six participants were randomly assigned post-HCT to receive gilteritinib or placebo. Although RFS was higher in the gilteritinib arm, the difference was not statistically significant (hazard ratio [HR], 0.679 [95% CI, 0.459 to 1.005]; two-sided P = .0518). However, 50.5% of participants had MRD detectable pre- or post-HCT, and, in a prespecified subgroup analysis, gilteritinib was beneficial in this population (HR, 0.515 [95% CI, 0.316 to 0.838]; P = .0065). Those without detectable MRD showed no benefit (HR, 1.213 [95% CI, 0.616 to 2.387]; P = .575). CONCLUSION Although the overall improvement in RFS was not statistically significant, RFS was higher for participants with detectable FLT3-ITD MRD pre- or post-HCT who received gilteritinib treatment. To our knowledge, these data are among the first to support the effectiveness of MRD-based post-HCT therapy.

Background: BMT CTN 1506/MORPHO was a randomized study of the FLT3 inhibitor gilteritinib versus placebo as post-transplant (HCT) maintenance therapy for patients (pts) with FLT3-ITD acute myeloid leukemia (AML). Patients with FLT3-ITD AML in first remission underwent HCT and then were randomized, in double-blind fashion, to either gilteritinib or placebo for 24 months. The primary endpoint was relapse-free survival (RFS), and a pre-specified secondary endpoint was the effect of measurable residual disease (MRD) on survival as detected by a highly sensitive assay for FLT3-ITD mutations. In the primary analysis, while RFS was higher for pts randomized to gilteritinib, the difference did not reach the pre-determined threshold for significance (HR: 0.679; 95% CI: 0.459, 1.005; 2-sided p-value: 0.0518). However, in the secondary analysis, the 50.6% of pts who had MRD detectable pre- or post-HCT had significantly higher RFS with gilteritinib (HR of relapse or death=0.515, 95% CI: 0.316, 0.838, p = 0.0065). In this post-hoc analysis, we examined 1) the impact on RFS of different levels of FLT3-ITD variant allele frequency (VAF) pre-randomization (immediately before or after HCT but prior to randomization to gilteritinib or placebo); 2) the impact of the presence of multiple mutations detected as MRD pre-HCT; and 3) the eradication of FLT3-ITD clones detected post-HCT during follow up on gilteritinib or placebo. Methods: First-pull marrow aspirates were collected from pts at two time points pre-randomization (pre-HCT and between 30-90 days post-HCT), as well as at 3, 6, 12, 18, and 24 months post-randomization. For MRD detection (performed at Invivoscribe; San Diego, CA), 700 ng input DNA was amplified by polymerase chain-reaction (PCR) using 25 cycles and primers flanking exons 14 and 15 of FLT3, followed by next-generation sequencing (NGS) analysis of the amplicons. Variant allele frequency (VAF) was calculated as FLT3 mutant reads/total FLT3 reads. For pts with multiple FLT3-ITD mutations, the VAF used in analysis was the sum of the VAFs for each FLT3-ITD variant. The lower limit of blank (LOB) of the assay is estimated to be 1 x 10 -6 VAF. For terminology, VAF > 1 x 10 -6 and < 1 x 10 -5 is referred to as MRD6, VAF > 1 x 10 -5 and < 1 x 10 -4 is MRD5, VAF > 1 x 10 -4 or greater is MRD4, and MRD0 equals no detectable MRD. Results: MRD was evaluated in 350/356 (98.3%) pts pre-HCT and 347/356 (97.5%) pts post-HCT. MRD was detected in 46% of pts pre-HCT and 19.9% prior to randomization, including 4.5% who did not have detectable MRD pre-HCT. The variant allele frequency (VAF) of detectable MRD ranged from a low of 1.09 x 10 -6 to a high of 3.0 x 10 -1. More than a single mutation was detected in 51 pts pre-HCT. Most of these 51 pts had 2 clones, but the total ranged from 2-9. The presence of more than one mutation detected as MRD immediately pre-HCT was associated with worse RFS compared with all other pts (MRD-positive with only one clone and MRD-negative), irrespective of treatment arm (Figure 1A; HR=2.428, 95% CI: 1.555, 3.792, P < 0.001). There was no significant difference in age, mutation length, VAF, or karyotype between pts with more than one mutation and those with single mutations. However, if the VAFs of pts with multiple FLT3-ITD mutations were summed and treated as a single value, then the median VAF for pts with multiple mutations was higher than for those with single mutations (2.14 x 10 -4 vs. 3.12 x 10 -5). The VAF (including the summed VAFs of pts with multiple mutations) at multiple levels correlated very closely with RFS after HCT. To illustrate this quantitative effect of peri-HCT FLT3-ITD VAF on RFS, Kaplan-Meier analysis of pts on the placebo arm grouped by MRD level is displayed in Figure 1B. In the 71 pts with MRD detectable post-HCT, the MRD was eradicated in 69% of pts on gilteritinib versus 44% of pts on placebo. Conclusions Pts with multiple FLT3-ITD mutations have a worse prognosis, which may simply be a reflection of increased disease burden (VAF). The quantity of FLT3-ITD MRD appears to highly correlate with outcome. Gilteritinib appears to augment the effect of HCT, as evidenced by increased eradication of MRD post-HCT. These data illustrate the potential utility of the PCR-NGS FLT3-ITD MRD assay in the management of pts with FLT3-ITD AML.

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal expansion of myeloid blasts in the peripheral blood, bone marrow, and/or other tissues. It is the most common form of acute leukemia among adults and accounts for the largest number of annual deaths from leukemias in the United States. Like AML, blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a myeloid malignancy. It is a rare malignancy characterized by the aggressive proliferation of precursors of plasmacytoid dendritic cells that frequently involves the bone marrow, skin, central nervous system, and other organs and tissues. This discussion section focuses on the diagnosis and management of BPDCN as outlined in the NCCN Guidelines for AML.

Abstract Overexpression of B-cell leukemia/lymphoma 2 (BCL2) renders acute myeloid leukemia (AML) cells resistant to chemotherapy and has been associated with unfavorable outcomes. Oblimersen (G3139) is a phosphorothioate 18-mer antisense oligonucleotide directed against the first 6 BCL2 codons. In a phase 1 study of AML patients treated with G3139, cytarabine, and daunorubicin induction with cytarabine consolidation, no antisense-related toxicity was reported, and BCL2 downregulation occurred in patients achieving complete remission. In this phase 3 trial, untreated older AML patients were randomized to cytarabine (100 mg/m2 per day on days 4-10) and daunorubicin (60 mg/m2 per day on days 4-6) followed by cytarabine consolidation (2000 mg/m2 per day on days 4-8) with (arm A) or without (arm B) G3139 (7 mg/m2 per day on days 1-10 [induction] or days 1-8 [consolidation]). A total of 506 patients were enrolled. No differences in toxicity were observed between arms. Estimated overall survival (OS) at 1 year was 43% for arm A and 40% for arm B (1-sided log rank P = .13), with no differences in disease-free (DFS; P = .26) or event-free survival (P = .80). Subgroup analyses showed patients age <70 years in arm A had improved OS by 1 month vs those in arm B (P = .04), and patients with secondary AML in arm A had better DFS vs those in arm B (P = .04). We conclude that addition of G3139 to chemotherapy failed to improve outcomes of older AML patients. However, more effective means of inhibiting BCL2 are showing promising results in combination with chemotherapy in AML. This trial was registered at www.clinicaltrials.gov as #NCT00085124.

Abstract Patients with core-binding factor (CBF) acute myeloid leukemia (AML), caused by either t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22), have higher complete remission rates and longer survival than patients with other subtypes of AML. However, ∼40% of patients relapse, and the literature suggests that patients with inv(16) fare differently from those with t(8;21). We retrospectively analyzed 537 patients with CBF-AML, focusing on additional cytogenetic aberrations to examine their impact on clinical outcomes. Trisomies of chromosomes 8, 21, or 22 were significantly more common in patients with inv(16)/t(16;16): 16% vs 7%, 6% vs 0%, and 17% vs 0%, respectively. In contrast, del(9q) and loss of a sex chromosome were more frequent in patients with t(8;21): 15% vs 0.4% for del(9q), 37% vs 0% for loss of X in females, and 44% vs 5% for loss of Y in males. Hyperdiploidy was more frequent in patients with inv(16) (25% vs 9%, whereas hypodiploidy was more frequent in patients with t(8;21) (37% vs 3%. In multivariable analyses (adjusted for age, white blood counts at diagnosis, and KIT mutation status), trisomy 8 was associated with improved overall survival (OS) in inv(16), whereas the presence of other chromosomal abnormalities (not trisomy 8) was associated with decreased OS. In patients with t(8;21), hypodiploidy was associated with improved disease-free survival; hyperdiploidy and del(9q) were associated with improved OS. KIT mutation (either positive or not tested, compared with negative) conferred poor prognoses in univariate analysis only in patients with t(8;21).