Single-Tube Flow Cytometry 12-Color Combination for the Screening, Classification, and Staging of Chronic Myelomonocytic Leukemia. Correlation with NGS Molecular Data
Authors: Piñero Paula , Gonzalez Rocio , Diaz Vanesa , Marin Elena , Gonzalez Marina , Lopez Javier , Manresa Pablo , Garcia Hernandez M. Carmen , Tarin Fabian 
Published: December 2024

Introduction:
Chronic Myelomonocytic Leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm with a great variable clinical presentation, evolution, and prognosis. The latest WHO revision has significantly updated criteria for the diagnosis and classification of CMML, highlighting the importance of an integrated phenotype, cytogenetic, and molecular study to achieve diagnosis and prognostic stratification. However, the access to advanced diagnostic techniques, such as Next-Generation Sequencing (NGS), is limited in many centers due to costs and complexity in the interpretation of molecular data.
Aims: To validate a Flow Cytometry 12-Color combination for the accurate diagnosis, characterization, and classification of CMML. To correlate phenotypic and molecular data with the different subtypes and stages of the disease. To discuss the potential advantages of applying the described strategy in daily practice from a cost-benefit perspective.
Methods:
Patients and Samples: We analyzed peripheral blood (PB) and bone marrow (BM) samples from 20 healthy controls and 77 patients with monocytosis (≥500/µL) using FCM and Next Generation Sequencing (NGS) protocols as described below.
The FCM single-tube combination was designed to be applicable to both PB and BM samples. The 12-markers included in the panel were HLADR-V450, CD45-V500, SLAN-FITC, CD64-PE, CD34-PerCP-Cy5.5, CD117-PECy7, CD300e-APC, CD14-APCH7, CD56-APCR178, CD2-BV605, CD16-BV786 and CD123-BV711. Samples were acquired using a FACSLyric cytometer (BD Biosciences). FCS files were analyzed using Infinicyt Software V 2.0 (Cytognos,). The analysis strategy was focused on granulocytic, monocytic, and dendritic cell compartments.
We performed an NGS panel study according to the international guide recommendations. NGS libraries were synthesized using the QIAseq Targeted Human Myeloid Neoplasms DNA Panel DHS-003Z.Data analysis was performed using CLC Genomics Workbench and the QCInterpret platform.
The study received approval from Alicante's University Hospital ethical committee, and all participants provided informed consent in accordance with the Declaration of Helsinki.
Results: We defined abnormal monocytic partition based on the distribution of monocytes subpopulations (classical (MO-1), inflammatory (MO-2) and non-classical (MO-3)) attending to the expression of CD14, CD16, and SLAN.According to the monocytic partition we differentiate 3 groups: patients with isolated monocytosis, patients with monocytosis+abnormal partitioning and CMML patients All patients with monocytosis and abnormal partition (n=11) demonstrated pathogenic mutations, mainly involving TET2 and SRSF2 genes (VAF 23-51%). In contrast, patients with normal partitioning (n=19), as the healthy controls, did not exhibited pathogenic mutations in any of the genes studied. Patients meeting CMML criteria exhibited a significant decrease in the MO-3 subpopulation (CD14dim, CD16+, SLAN+) compared to the healthy controls (p&lt;0.001). The most frequent mutations involved TET2, ASXL1, SRSF2, ZRSR2 and RAS genes. Patients with proliferative CMML more commonly harbored NRAS and KRAS mutations, along with an inflammatory immunophenotype (CD16+SLAN-), compared to those with myelodysplastic CMML. We defined the percentage of blasts cells (myeloblasts + monoblasts + promonocytes) based on the expression of CD34, CD117, CD14 and CD300e in order to classify patients in CMML types 1 and 2. Patients classified as CMML-2 by phenotypic criteria presented mutations in ASXL1 (p=0.03), RUNX1 (p&lt;0.01), and KRAS (p&lt;0.01), as well as a higher frequency of CD56 expression and higher-risk molecular profiles (CPSS-mol score, p&lt;0.01) compared to CMML-1. This association was not observed when the staging was based on morphological criteria.
Conclusions Clonal monocytosis and CMML exhibit a distinctive immunophenotype and mutational profile, which are crucial for their differential diagnosis. Enumerating blasts cells based on their phenotypic characteristics is closely linked to the clonal architecture of CMML and provides an objective tool for disease staging, outperforming traditional morphology-based methods. The proposed FCM panel offers an easy, cost-effective and fast approach for the diagnosis and stratification of CMML, saving time and efforts for younger patients requiring critical therapeutic decisions.