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Introduction
The CCAAT/enhancer-binding protein alpha (CEBPA) is a transcription factor essential for the differentiation of granulocytes. The expression of transcription factor CEBPA initiates at the time of dedication of stem cells to myeloid lineage, but it is specifically upregulated in granulocytes and is downregulated in mature peripheral blood monocytes[1]. It is well demonstrated that in the presence of defective CEPBA, only myelocytic lineages are affected while other hematopoietic lineages remain as usual.
Leukemia develops from the serial possession of somatic mutations in hematopoietic stem and ancestor cells with the capacity to self-replicate and propagate the neoplastic clone.[2,3] Although some mutations, such as those in DNMT3A, TET2, and ASXL1, are more common in clonal haematopoiesis and appear to be fairly early events in leukemogenesis, others tend to be acquired later in the course of leukemia development, including mutations in FLT3, NRAS, and RUNX1. The combinations of mutations that eventually drive leukemogenesis are influenced by biological cooperativity and mutual exclusivity between mutated genes. Due to their overriding impact on disease phenotype and disease outcome, genetic aberrations are given priority in defining acute myeloid leukemia (AML) disease classification. The classification system of myeloid malignancies that recently appeared is hereby named the International Consensus Classification (ICC) [table 1] and the fifth edition of the World Health Organization (WHO) classification (WHO-HAEM5) [table 2].[4,5] Since 2017, new data has surfaced that illuminates the need to acclimate the risk classification. In addition to baseline genetic characterization, the significance of response to initial therapy and assessment of early MRD in individual risk assignment is stressed.[3,6] For further backing, an ELN AML risk classification has been developed based on data from intensively treated patients [table 3].[7]
International Consensus Classification (ICC) 2022 of AML.
ICC 2022
AML with recurrent genetic abnormalities (requiring equal or greater than 10% blasts, except*)
AML with t(8;21)(q22;q22.1)/RUNX1::RUNX1T1
AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22)/CBFB::MYH11
Acute promyelocytic leukemia (APL) with t(15;17) (q24.1;q21.2)/PML::RARA; APL with other RARA rearrangements
AML with t(9;11)(p21.3;q23.3)/MLLT3::KMT2A; AML with other KMT2A rearrangements
AML with t(6;9)(p22.3;q34.1)/DEK::NUP214
AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2)/GATA2::MECOM(EVI1); AML with other MECOM rearrangements
AML with BCR::ABL1 fusion*
AML with other rare recurring translocations
AML with mutated NPM1
AML with in-frame bZIP CEBPA mutations
AML requiring equal or greater than 20% blasts**
AML with mutated TP53 (VAF >10%)
AML with myelodysplasia-related gene mutations
AML with myelodysplasia-related cytogenetic abnormalities
Therapy-related myeloid neoplasms
NA (BECOMING DIAGNOSTIC QUALIFIER)
AML not otherwise specified; subtyping optional
AML with minimal differentiation
AML without maturation
AML with maturation
Acute myelomonocytic leukemia
Acute monoblastic/monocytic leukemia
Pure erythroid leukemia***
Acute megakaryoblastic leukemia
Acute basophilic leukemia
Myeloid sarcoma
WHO classification (WHO-HAEM5) of AML.
WHO 2022
AML with defining genetic abnormalities (no blast % cut-off, except*)
AML with RUNX1::RUNX1T1 fusion
AML with CBFB::MYH11 fusion
Acute promyelocytic leukemia with PML::RARA fusion
AML with KMT2A rearrangement
AML with DEK::NUP214 fusion
AML with MECOM rearrangement
AML with RBM15::MRTFA fusion
AML with BCR::ABL1 fusion*
AML with NUP98 rearrangement
AML with other (rare) defined genetic alterations*
AML with NPM1 mutation
AML with CEBPA mutation*#
AML requiring equal or greater than 20% blasts
AML, myelodysplasia-related
Therapy-related myeloid neoplasms
NA (BECOMING NEW ENTITY OF SECONDARY MYELOID NEOPLAMS)
Frequencies, response rates, and outcome measures should be reported by risk category, and, if sufficient numbers are available, by specific genetic lesions indicated.
Mainly based on results observed in intensively treated patients. Initial risk assignment may change during the treatment course based on the results from analyses of measurable residual disease.
Concurrent KIT and/or FLT3 gene mutation does not alter risk categorization.
AML with NPM1 mutation and adverse risk cytogenetic abnormalities are categorized as adverse-risk.
Only in-frame mutations affecting the basic leucine zipper (bZIP) region of CEBPA, irrespective of whether they occur as monoallelic or biallelic mutations, have been associated with favorable outcome.
The presence of t(9;11)(p21.3;q23.3) takes precedence over rare, concurrent adverse risk gene mutations.
Excluding KMT2A partial tandem duplication (PTD).
Complex karyotype: ≥3 unrelated chromosome abnormalities in the absence of other class-defining recurring genetic abnormalities; excludes hyperdiploid karyotypes with three or more trisomies (or polysomies) without structural abnormalities.
Monosomal karyotype: presence of two or more distinct monosomies (excluding loss of X or Y), or one single autosomal monosomy in combination with at least one structural chromosome abnormality (excluding core-binding factor AML).
For the time being, these markers should not be used as an adverse prognostic marker if they co-occur with favorable-risk AML subtypes.
TP53 mutation at a variant allele fraction of at least 10%, irrespective of the TP53 allelic status (mono- or biallelic mutation); TP53 mutations are significantly associated with AML with complex and monosomal karyotype
Genetic abnormalities are the most potent prognostic markers for AML. In recent times, mutated genes in AML have been assessed along with traditional chromosomal analysis, which helps in the invention of more advanced targeted therapies. One of the most important prognostic genes for AML is CEPBA, and its mutation (CEBPAmu) is present in almost 10%–15% of de novo AML cases. One-third of CEBPAmu is single-mutated CEPBA (CEBPAsm), a heterozygous monoallelic mutation, and two thirds are double-mutated CEBPA (CEBPAdm), mostly biallelic N- and C-terminal mutations. However, CEBPAsm has a poorer prognosis than CEBPAdm; thus, the utility of CEBPAsm as a prognostic factor has not been clarified.[8,9,10,11,12] Though it is not certified, CEBPAsm has a poorer prognosis than CEBPAdm.[13]
CEPBA messenger RNA has two translation initiation sites and two isoforms: p42 (42 kDa) and p30 (30 kDa). The basic leucine zipper (bZIP) domain located on the C-terminal is common to both isoforms and it helps in DNA binding to the major groove of the DNA molecule by protein dimerization. One-third of CEBPAsm is in mutation in the bZIP domain, whereas the remaining are out of the bZIP domain. In contrast, mutations in CEBPAdm can involve both bZIP domains, one of them, or none.
This study mainly focuses on the effect of the bZIP domain mutation of CEBPAmu on the prognosis of AML.
Materials and methods
Sources of data and inclusion criteria
We searched PubMed, Google Scholar, and EMBASE for studies related to our study that were published after September 30, 2021. Synonyms used for the search process were acute myeloid leukemia, CEBPA, mutation, prognosis. The search was confined to human studies without language limitations. We did a manual search of reference lists in applicable studies to find out the applicability of the information to our study. We considered the study if it met the following criteria: (i) published after September 30, 2021; (ii) studies considering patients diagnosed with AML; (iii) studies pointing to the CEBPA mutation in bZIP domain; and (iv) studies having survival information of the patients.
Exclusion criteria
Studies did not consider any particular treatment protocol and did not give information on prognosis.
Data analysis
The review team discussed the inclusion criteria for studies and extracted data independently from articles. Data were double checked before they were put into statistical analysis to count out any discrepancies. Data excluded from papers were name of first author, time of publication, number of subjects, age group, prevalence of CEBPA mutations, therapeutic strategy, and complete remission (CR).
Statistical analysis
The Kaplan–Meier method was used to estimate overall survival (OS), disease-free survival (DFS), and relapse rate (RR) in those concerned studies. The authors of this study did not perform any additional Kaplan–Meier method. OS was defined as the time from study entry to death or date of last follow-up in surviving patients; DFS was defined as the time from end of course 1 for patients in CR until relapse or death or date of last follow-up for those without an event.[14] These above-mentioned definitions of OS and DFS were stated by original studies. In addition, definition of end point was uniform across three studies. For the rest of the analysis, authors used Statistical Package for the Social Sciences (SPSS) 2023. To indicate statistical significance, a 5% level of probability was used.
Results
Three studies comprising 8694 patients were included in our study[15,16,17]. Studies included in our study originated from Japan, Germany, and Seatle (USA). We considered every age group for our study.
CEBPA mutations and their frequency
The frequency of all CEBPAmu is shown in Figure 1. There were 59 patients with CEBPAsm and 103 patients with CEBPAdm among the total 1028 patients in the study conducted by Wakita.[15] Of the patients with CEBPAdm, 91.3% (94 of 103) had a combination of mutations in the bZIP domain (CEBPAmu in bZIP) and mutations out of the bZIP domain (i.e., CEBPAmu out of bZIP), 2.9% (3 of 103) of the patients had two CEBPAmu in bZIP, and 5.8% (6 of 103) of the patients had two CEBPAmu out of bZIP. Furthermore, 32.2% (19 of 59) of the patients with CEBPAsm had mutations in the bZIP domain (CEBPAsm in bZIP) and 67.8% (40 of 59) of patients had mutations out of the bZIP domain (CEBPAsm out of bZIP).
Figure 1:
Frequency distribution of mutations reported in studies separated on the basis of author’s name.
A total of 240 of the 4708 patients, which corresponds to 5.1% of the total patients, were positive for the CEBPA mutation in the study conducted by Taube.[16] In addition, 131 of 240 patients (54.6%) showed two CEBPA mutations mostly associated with combinations of TAD and bZIP mutations, mentioned as CEBPAbi and CEBPAsm mutations, respectively, found in 109 out of 240 patients (45.4%), most of which were associated with independent mutations of TAD and bZIP.
Mutations in the bZIP domain were identified in 160 (5.4%) of 2958 patients. Among patients with bZIP mutations, 132 (82.5%) harbored a cooperating TAD domain mutation on the other allele and the remaining 28 patients (17.5%) lacked a second CEBPA mutation.[17]
Figure 2 shows the frequency of CEBPAmu mutations in the three studies considered in our study.
Figure 2:
Comparison of percentage frequency of CEBPAmu found out of total study population.
Clinical correlation of AML with CEBPAmu in bZIP
Most of the patients with AML with CEBPAmu on bZIP who were studied in the studies mentioned above belonged to immediate-risk chromosomal classification. Compared to patients bearing CEBPA WT mutation, those with CEBPA biallelic mutation were significantly younger (median age, 46 years) at diagnosis, similar to those with CEBPAsm on bZIP (median age, 50 years), whereas patients with CEBPAsm on TAD were significantly older (median age, 63 years) and were more comparable to the CEBPA WT mutation group (median, 57 years).
Comparison between CEBPAsm in bZIP and CEBPAsm out of bZIP
FLT3–ITD mutations were detected in 32.5% (13 of 40) of patients in the CEBPAsm out-of-bZIP group, which was higher than the 5.3% (1 of 19) of patients in the CEBPAsm in-bZIP group, and NPM1 mutations were detected in 35.0% (14 of 40) of patients in the CEBPAsm out-of-bZIP group and 26.3% (5 of 19) of patients in the CEBPAsm in-bZIP group according to the study conducted by Wakita et al.[15]
Based on the targeted NGS approach, additional mutations were identified in 208 of 240 patients with a CEBPA mutation (86.7%). The most frequently mutated genes were TET2 (70 of 240; 29.2%), GATA2 (68 of 240; 28.3%), DNMT3A (45 of 240; 18.8%), FLT3–ITD (39 of 240; 16.3%), NPM1 and NRAS (31 of 240; 12.9% each), and WT1 (30 of 240; 12.5%), according to the study by Taube et al.[16]
CEBPA mutations and outcome
Multivariate analyses for OS and cumulative incidence of relapse (CIR) were conducted on patients in a study conducted by Wakita et al., and they subsequently found OS (hazard ratio of 0.3287) and multivariate analysis for CIR in 525 patients who achieved CR showed that CEBPAmu in bZIP was an independent favorable prognostic factor of the CIR hazard ratio of 0.6157. A study conducted by Taube found that the CEBPA biallelic mutation had a significantly higher rate of CR as well as a higher median OS (CEBPA biallelic, 103.2 months; CEBPAsm, 21.9 months). Patients carrying CEBPAsm on bZIP showed significantly higher CR, with a remission rate close to 86% (Figure 3). Another study done by Tarlock presented the OS of the CEBPA bZIP mutation as 89% (Figure 4) and the OS of CEBPAdm as 81%.[17]
Figure 3:
Complete remission (CR) rate percentage in the three studies.
Figure 4:
Overall survival (OS) rate in percentage in the three studies, showing favorable prognosis.
Discussion
We analyzed 8694 patients in a combination of three studies by Wakita, Taube, and Tarlock and double checked all the data extracted from those studies.
In the study conducted by Wakita et al., CEBPAdm was found to be a favorable prognostic marker. In addition, it was seen that if a single mutation is seen in CEBPA in the bZIP domain or in the bZIP domain, a mutation in the single or double position in the case of CEBPAdm is markedly favorable for diagnosis. CEBPAdm, which has been recommended in many previous guidelines, was found to be a confounding factor in CEBPAmu in the bZIP group and was not obtained as an independent prognostic factor. They also speculated that altered bZIP function acquired by the CEBPA bZIP mutant determines the sensitivity of the disease to chemotherapy. This surely explains why CEBPAdm is a better prognostic factor than CEBPAsm if linked with the bZIP domain, though it still needs further discussion. They also found CEBPAmu-bZIP to have an overall survivability of 53% (Figure 4) and CR of 80%.
In the study done by Tarlock et al. in a large cohort of 2948 children and young adults with newly diagnosed AML, they demonstrated that patients with CEBPA mutations that had single bZIP domain mutations experienced outcomes nearly identical to those of patients with biallelic CEBPA mutations. Georgi et al. reported on a cohort of 4578 adult patients with AML and showed analogous outcomes for patients with CEBPA-dm and CEBPA-bZIP.[18] In this study, CR was noted in 22 of 28 patients with CEBPA-bZIP, which corresponds to 78.6%. It was also found that 64% of patients with the CEBPA bZIP mutation experienced 5-year event-free survival.
Taube et al. found that only 90% of patients with biallelic CEBPA mutations containing typical bZIP mutations show a better outcome, which indicates that only those should be assigned to the favorable risk group. A typical bZIP mutation was also found in 90% of biallelic CEBPA mutations.
