Multiparameter Flow Cytometry-Based Measurable Residual Disease Assessment in the Patients with CCAAT Enhancer-Binding Protein a Gene (CEBPA) Mutated Acute Myeloid Leukemia
Bibliographic record
Abstract
Introduction Acute myeloid leukemia (AML) with mutations in CCAAT enhancer-binding protein A gene (CEBPA) is a unique AML subtype with heterogeneous molecular features such as CEBPA biallelic or double mutations (CEBPAdm) and in-frame basic leucine zipper region (CEBPAinf-bZIP) mutations. CEBPAdm and CEBPAinf-bZIP mutations can be monitored by next-generation sequencing (NGS)-based residual disease (RD) monitoring but with limitation due to guanine-cytosine (GC) rich content of the CEBPA gene. Alternatively, multiparameter flow cytometry (MFC) can be used for RD assessment in these CEBPA mutated AML subtype. Patients and methods The study cohort consisted of patients diagnosed with AML at our institution between 2015 and 2022. NGS was conducted using a custom hybrid-capture NGS panel (Oxford Gene Technology, Kidlington, UK) targeting 49 myeloid genes regions including the entire consensus DNA sequence of CEBPA. Following sequencing (Illumina Miseq v2), CEBPA variants analysis included alignment to the GRCh37/hg19 human genome reference (Burrows-Wheeler Aligner v 0.7.12), base calling (GATK v3.3.0). and variant calling (Varscan v2.3.8), and the mean depth of coverage of the KMT2A exon interval was calculated using Picard v1.130. The analytical sensitivity of this NGS panel is 3-5% for the detection of small nucleotide variants as well as larger insertion deletion and duplications in CEBPA. The present study evaluated treatment outcomes in 150 patients with CEBPA mutated AML (CEBPA-AML) who achieved first complete remission (CR1), and compared the outcomes according to MFC-based RD status at CR1 assessment. Presentation marrow aspirate samples were screened for leukemia-associated phenotypes (LAIP) using 10 color flow. RD assessment has been a standard of care in our institution since 2015 based on the LAIP and/or “different from normal” (DfN), which was performed in parallel with morphologic assessment at CR1 after induction chemotherapy. The limit of detection (LOD) of the RD assay was 0.1%. Relapse-free survival (RFS) was calculated from CR1 to the date of relapse or death from any cause. Overall survival (OS) was calculated from diagnosis to the date of death. Cumulative incidence of relapse (CIR) was calculated considering competing events. Cox's proportional hazard and Fine-gray model were appropriately applied for univariate and multivariate analysis. Results In total NGS detected 211 CEBPA variants in 150 patients. 44 patients CEBPAinf-bZIPwere determined based on the presence of an in-frame variant with the CEBPA bZIP domain. 37 patients were determined CEBPAdm due a CEBPAinf-bZIP combined to a truncating variant (frameshift, nonsense or splicing) in the N-terminal end of CEBPA. The remaining patients 106 patients although harboring a CEBPA variant could not be identified as CEBPAinf-bZIP or CEBPAdm. Based on NGS results, 150 pts were diagnosed with CEBPA-AML. According to the MRC 2010 risk group, 104 (69.4%) was stratified as intermediate risk, 25 (16.7%) as adverse and 6 (4.0%) as favorable. Out of 98 pts who received intensive chemotherapy, 84 pts (90.3%) achieved remission. Out of 74 pts in CR who has available MFC-RD result, 22 (29.7%) showed detectable MFC-RD (RDpos), while 52 pts (70.3%) showed undetectable RD (RDneg). With a median follow-up duration of 43 months, 20 pts (24%) died, 12 (14%) relapsed, and 29 (34%) underwent HCT in CR1 of whom 4 pts (4%) relapsed after HCT. At 2 years, the RDneg group showed a higher RFS (97.8%) and OS rate (86.7%) compared to the RDpos group (77.3%, p=0.06; 56.5%, p=0.009). With respect to CIR, the RDpos group showed higher CIR (20.3%) than the RDneg group (9.5%; p=0.10),. In the CEBPAb-Zip subgroup those with RDneg showed a higher OS rate at 2 years (91.6%) and lower CIR (3.6%) compared to the RDneg group (71.7%; p=0.006 for OS; 19.2%, p=0.05 for CIR). Multivariate analysis confirmed that MFC-RDneg at CR1 is a favorable prognostic factor in the overall population with respect to RFS (HR 0.43, p=0.04), OS (HR 0.399, p=0.032) and CIR (HR 0.39, p=0.09), particularly in the bZIP subgroup for RFS (HR 0.19, p=0.007), OS (HR 0.31, p=0.006) and CIR (HR 0.17, p=0.002). Conclusion These findings suggest that in CEBPA mutated AML, MFC-based RD assessment is feasible and will provide an excellent predictive measure when NGS-based RD assessment is limited. Further studies are warranted to reach a clearer conclusion with a larger number of replication cohorts.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".