CEBPA Mutations in 4708 Patients with Acute Myeloid Leukemia - Differential Impact of bZIP and TAD Mutations on Outcome

Biallelic mutations of the CEBPA gene (CEBPAbi) define a distinct entity associated with favorable prognosis, however the role of monoallelic mutations (CEBPAsm) is poorly understood. We retrospectively analyzed 4708 adult AML patients recruited into Study Alliance Leukemia trials to investigate the prognostic impact of CEBPAsm. CEBPA mutations were identified in 240 patients (5.1%), 131 CEBPAbi and 109 CEBPAsm (60 affecting the amino-terminal transactivation domains (CEBPAsmTAD) and 49 the carboxy-terminal DNA-binding or basic leucine zipper region (CEBPAsmbZIP)). Interestingly, CEBPAbi and CEBPAsmbZIP patients shared several clinical factors, i.e. were significantly younger (median 46 years and 50 years) and had higher WBC counts at diagnosis (median 23.7 and 35.7 109/l) compared to CEBPAsmTAD patients (median age 63 yrs., median WBC 13.1 109/l; p<.001). Co-mutations were also similar in both groups, e.g. GATA2 mutations (35.1% CEBPAbi; 36.7% CEBPAsmbZIP vs. 6.7% CEBPAsmTAD; p<.001) or NPM1 mutations (3.1% CEBPAbi; 8.2% CEBPAsmbZIP vs. 38.3% CEBPAsmTAD; p<.001). CEBPAbi and CEBPAsmbZIP, but not CEBPAsmTAD were associated with significantly improved overall (median OS: 103 and 63 vs. 13 months) and event-free survival (median EFS: 20.7 and 17.1 vs. 5.7 months), in univariate and multivariable analyses. More detailed analysis revealed that the clinical and molecular features as well as the favorable survival were confined to patients showing in-frame mutations in bZIP (CEBPAbZIP-inf). When grouping patients into CEBPAbZIP-inf and CEBPAother (including CEBPAsmTAD and other non-CEBPAbZIP-inf patients), only CEBPAbZIP-inf patients showed superior CR rates and the longest median OS and EFS, arguing for a previously undefined prognostic role of this type of mutations.

Frequency and Clinical Impact of WT1 Mutations in the Context of CEBPA-Mutated Acute Myeloid Leukemia

Introduction: Several studies have confirmed that mutations in the Wilms tumor 1 (WT1) gene occur in adult acute myeloid leukemia (AML). However, few data are available regarding the incidence of WT1 mutations in CEBPAmut AML and their impact. Methods: We retrospectively analyzed the frequency and clinical impact of WT1 mutations in 220 newly diagnosed AML patients with CEBPA mutations. Chromosome karyotype analysis was performed by R or G banding method and further confirmed either by fluorescence in situ hybridization (FISH) and/or by multiple reverse transcription polymerase chain reaction (multiple RT-PCR). Mutations were detected with a panel of 112 mutational genes using next-generation sequencing (NGS). FLT3-ITD, NPM1 and CEBPA mutation were detected by PCR Sanger sequencing.Results: Overall, 30 WT1 mutations were detected in 29 of the 220 patients (13.18%) screened. These mutations clustered overwhelmingly in exon 7 (16 mutations in 15 patients), but they were also detected in exon 8 (n=6) and exon 9 (n=8). WT1-mutated (WT1mut) patients presented with lower platelet counts (P=0.0481), higher WT1 expression (P=0.0371), and a negative trend with the M5 subtype (P=0.07) compared to WT wild-type (WTwt) patients. WT1 mutations were found to be significantly more frequent in AML patients with double-mutated CEBPA (CEBPAdm) than in AML patients with single-mutated CEBPA (P=0.043). Of note, the concomitant mutations in epigenetic regulators were inversely correlated with WT1 mutations (P=0.003). Patients with newly diagnosed WT1mut AML had inferior relapse-free survival, event-free survival and overall survival compared with patients with WT1wt AML (P=0.002, 0.004, and 0.010, respectively). Conclusion: Our data showed that WT1 mutations are frequently identified in CEBPAmut AML, especially in CEBPAdm AML. Patients with WT1 mutations show distinct clinical features, a spectrum of comutations, and poor prognosis compared to WT1 wild-type patients.

Outcomes of Therapy with Venetoclax Combined with Hypomethylating Agents in Favorable-Risk Acute Myeloid Leukemia (AML)

Introduction: Venetoclax (VEN) in combination with a hypomethylating agents (HMA) is associated with a high rate of composite remission (complete remission [CR] and complete remission with incomplete recovery [CRi]) among older and unfit patients with untreated AML. However, data regarding the activity of VEN-HMA in those patients with favorable-risk AML is limited, particularly in those with core-binding factor (CBF) alterations. Although more frequent among younger patients, favorable-risk alterations are also observed among older patients, often unfit for intensive regimens. Even among the subset of older patients (>60 years) with favorable-risk AML eligible for intensive regimens, long-term outcomes are poorer in comparison to younger patients. Methods: We retrospectively analyzed outcomes of 46 patients with favorable-risk AML who underwent therapy with VEN-HMA between 2016-2020 at 4 academic cancer centers in US. Favorable-risk AML was defined by the presence of either CBF [t(8;21) and inv(16) or t(16;16)], NPM1 mutation in the absence of FLT-3 ITD mutations; or bi-allelic CEBPA mutations. Results: Forty-six patients with favorable risk AML were treated with HMA-VEN, including 26 (57%) with newly diagnosed (ND) and 20 (43%) with relapsed/refractory (R/R) AML (Table1). Ten (22%) patients had CBF, 21 (46%) had NPM1 mutations (NPM1m), and 13 (28%) had bi-allelic CEBPA mutations (CEBPAm). The median age was 70 years, and 54% were females. Patients with R/R AML were younger than ND patients (56 vs. 72 yrs, p=0.003). Twenty (44%) patients had secondary or therapy-related AML, including half of ND patients. The median lines of prior therapy were 2(1-4) in patients with R/R AML, including 6 (30%) who had failed prior allogeneic HCT. Eleven (24%) patients had received HMA prior to HMA-VEN therapy, including 1 patient in the ND cohort for prior MDS. Eleven (24%) patients received azacitidine in combination with VEN, while the rest (76%) of patients received decitabine, including 14 patients who received 10-day decitabine during the first cycle. The CR/CRi rate among the whole cohort was 80%, including 52% CR and 28% CRi. There was no statistically significant difference in CR/CRi rate between ND and R/R patients (88% vs. 70%, P =0.15). However, patients with history of prior HMA exposure had lower response rate compared to HMA-naïve patients (55% vs. 88%, p= 0.025). No difference in response was observed based on the favorable genetic alteration subgroups (80% in CBF vs. 86% in NPM1m vs. 77% in CEBPAm, p=0.44). Furthermore, no difference in response was observed according to patient age (p= 0.83), AML types (de novo vs. secondary; p= 0.47), prior transplant (p=1.00), or the type and schedule of HMA (P=0.66). Among the responders who had MRD assessment done (n= 26), 22 (85%) achieved MRD negativity by multicolor flow cytometry. Post response, 13 (35%) patients underwent allogeneic transplant consolidation. The median overall survival (OS) for the whole cohort was 18 months (12.5-NA). Median leukemia-free survival (LFS) was 13.2 months (7-20.2) for all responders, 11.2 months (1.7-NA) for ND responders, and 14.0 months (1-NA) for RR responders (p=0.986). The 30- and 60-day mortality for the whole cohort was 0% and 9%, respectively. Conclusion: In patients with favorable-risk AML, VEN-HMA combination is associated with a highly promising CR/CRi rate, with durable responses. The majority of responders achieved MRD negativity. Patients with prior use of HMA had lower response rate with VEN-HMA, nonetheless, over half of these patients responded despite most being treated in the R/R setting. Disclosures Pullarkat: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genetech: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie, Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Dova: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Stein:Stemline: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Marcucci:Abbvie: Speakers Bureau; Merck: Other: Research Support (Investigation Initiated Clinical Trial); Novartis: Speakers Bureau; Pfizer: Other: Research Support (Investigation Initiated Clinical Trial); Takeda: Other: Research Support (Investigation Initiated Clinical Trial); Iaso Bio: Membership on an entity's Board of Directors or advisory committees. Yaghmour:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Alexion: Consultancy, Speakers Bureau. Bhatt:Omeros: Consultancy; Agios: Consultancy; Rigel: Consultancy; Tolero: Research Funding; Pfizer: Research Funding; Abbvie: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Partnership for health analytic research: Consultancy; Takeda: Consultancy; Jazz: Research Funding; National Marrow Donor Program: Research Funding; Oncoceutics: Other. Fathi:Takeda: Consultancy, Research Funding; Jazz: Consultancy; Forty Seven: Consultancy; Daiichi Sankyo: Consultancy; Amphivena: Consultancy; Blueprint: Consultancy; Kura Oncology: Consultancy; Boston Biomedical: Consultancy; Astellas: Consultancy; Trovagene: Consultancy; Novartis: Consultancy; PTC Therapeutics: Consultancy; Agios: Consultancy, Research Funding; BMS/Celgene: Consultancy, Research Funding; Kite: Consultancy; Pfizer: Consultancy; Trillium: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy, Research Funding; Abbvie: Consultancy; Newlink Genetics: Consultancy.

Two novel CEBPA mutations in a Turkish patient with acute myeloid leukemia

Acute myeloid leukemia (AML) was first categorized in 1976 by French, American and British researchers, and divided into eight subgroups (M0 to M7), depending on the cytochemical or histological changes in the leukemic cells. The gene mutations of FLT3-ITD, CEBPA and NPM1 are the most common that cooperate together in the prognosis of AML. The CEBPA gene that is a hematopoietic transcription factor, is located on chromosome 19q13.11, and its prevalence is between 5.0 and 14.0% in AML. The patient was referred to our clinic suffering from menorrhagia, unplanned weight loss in a month and low platelet levels, and was diagnosed with AML on clinical and laboratory examination. Here, we report a patient carrying two novel pathogenic mutations that create a frameshift mutation on the CEBPA gene, c.940_941insCCGTCG TGGAGACGA CGAAGG and c.221_222delAC by Sanger sequencing methodology.

Nanopore Targeted Sequencing for Rapid Gene Mutations Detection in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) clinical settings cannot do without molecular testing to confirm or rule out predictive biomarkers for prognostic stratification, in order to initiate or withhold targeted therapy. Next generation sequencing offers the advantage of the simultaneous investigation of numerous genes, but these methods remain expensive and time consuming. In this context, we present a nanopore-based assay for rapid (24 h) sequencing of six genes (NPM1, FLT3, CEBPA, TP53, IDH1 and IDH2) that are recurrently mutated in AML. The study included 22 AML patients at diagnosis; all data were compared with the results of S5 sequencing, and discordant variants were validated by Sanger sequencing. Nanopore approach showed substantial advantages in terms of speed and low cost. Furthermore, the ability to generate long reads allows a more accurate detection of longer FLT3 internal tandem duplications and phasing double CEBPA mutations. In conclusion, we propose a cheap, rapid workflow that can potentially enable all basic molecular biology laboratories to perform detailed targeted gene sequencing analysis in AML patients, in order to define their prognosis and the appropriate treatment.

Myeloid lineage enhancers drive oncogene synergy in CEBPA/CSF3R mutant acute myeloid leukemia

Acute Myeloid Leukemia (AML) develops due to the acquisition of mutations from multiple functional classes. Here, we demonstrate that activating mutations in the granulocyte colony stimulating factor receptor (CSF3R), cooperate with loss of function mutations in the transcription factor CEBPA to promote acute leukemia development. The interaction between these distinct classes of mutations occurs at the level of myeloid lineage enhancers where mutant CEBPA prevents activation of a subset of differentiation associated enhancers. To confirm this enhancer-dependent mechanism, we demonstrate that CEBPA mutations must occur as the initial event in AML initiation. This improved mechanistic understanding will facilitate therapeutic development targeting the intersection of oncogene cooperativity.