P1011: novel 12-color fcm + ngs strategy for the accurate diagnosis and stratification of chronic myelomonocytic leukemia. experience from the hematological diagnosis group of alicante.

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Chronic myelomonocytic leukemia (CMML) is a myelodysplastic-mieloproliferative neoplasm of heterogeneous presentation. The new WHO classification enhances the importance of integrating phenotypic, cytogenetic and molecular studies for the diagnosis and stratification of CMML. To face this challenge, the diagnostic units should proceed to a rational screening of monocytosis optimizing the use of the new available techniques. Aims: 1.Validation of a diagnosis protocol; based on the sequential use of 12-colors flow cytometry (FCM), FISH and NGS for the characterization and stratification of CMML. 2.To explore the diagnostic and prognosis value of the phenotypic and molecular findings in the different CMML subtypes. Methods: • Prospective study of peripheral blood (PB) and bone marrow (BM) samples from 62 CMML patients (diagnosed between 2020 -2023). • Immunophenotypic characterization of patients using a 12-colors FCM single tube combination (HLADR, CD45, SLAN, CD64, CD34, CD117, CD300e, CD14, CD56, CD2, CD16, CD123) using multidimensional analysis strategies. • NGS study using the Qiaseq targeted DNA human myeloid Neoplasms panel DHS-003Z (Qiagen). The sequencing process was performed in a Nextseq 500 sequencer (illumina). • Two different softwares were used for the analysis of the NGS results: CLC Genomics Workbench and QCI Interpret (Qiagen). Results: 1-The proposed 12-colors FCM combination allows the analysis of monocytes distribution [classical (CD14+/CD16−/SLAN-), intermediate (CD14+/CD16+/SLAN-) and non-classical (CD14low/CD16+/SLAN+)] as well as their phenotypic characterization. 2-CMML patients showed immunophenotypic features indicative of monocytic compartmentalization (>94% classical monocytes or <1.7% non-classical monocytes CD14+ SLAN+) with/without aberrant markers). The 59% of patients corresponded to LMMC-1 and the 41% to CMML-2. (Figure 1) 3-Virtually all the patients with phenotypic criteria presented clinically relevant mutations, the most frequently altered genes were TET2, ASXL1 and SRSF2 (Figure 2-A). The 90% of patients carried alterations in at least one of these 3 genes and the 75% showed alterations in 2 or more. 4-In addition, the vast majority of patients (90%) presented mutations of uncertain significance. These alterations especially affected to TET2 or ZRSR2 and EZH2 or DNMT3A, although, to a lesser extent (Table 1). 5-Interestingly, TET2 mutations were frequently multiple (2-5 mutations/patient), affecting different positions at the gene. Alterations consisted of single nucleotide variants (SNVs) and small deletions. This observation was significatively more frequent than what has been described for myelodysplastic syndromes and myeloproliferative patients. 6-In ASXL1, the 80% of alterations corresponded to a recurrent mutation (c.1934dupG). All the cases with alterations in SRSF2, showed a SNV at the position c.284 (C>T, C>A, C>G). Finally, the finding of a constant alteration (c.1403 G>A) in ZRSR2 was detected in a 30% of patients. 7-Finally, mutations in NRAS, RUNX1 and p53 were more frequently observed in patients with CMML-2. While the presence of multiple mutations in TET2 were related to CMML-1. (Table 2). Summary/Conclusion: CMML displays a recognizable immunophenotype and a characteristic mutational landscape, both of them are very useful for its diagnosis and stratification. A significant proportion of the uncertain significance mutations were recurrent, suggesting their potential involvement in the disease, as well as their usefulness in clinical practice. The described methodology has allowed to validate an integrated diagnostic strategy, maximizing the potential of the available techniques.Keywords: Flow cytometry, TET2, Gene mutation, CMML