ELN IMDS Flow Working Group Validation of the Monocyte Assay for Chronic Myelomonocytic Leukemia Diagnosis By Flow Cytometry

Abstract

Introduction It was proposed that peripheral blood (PB) monocyte subset analysis evaluated by flow cytometry, hereafter referred to as "monocyte assay", could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay required a large multicenter validation. Methods PB and/or bone marrow (BM) samples from adult patients displaying monocytosis were assessed with the "monocyte assay" by ten ELN iMDS Flow working group centers (6 equipped with BD FACSCanto™ II (BD Biosciences), 3 with Navios™ (Beckman Coulter) and one with BD™ LSRII (BD Biosciences)) with harmonized protocols. The corresponding files were reanalyzed in a blind fashion by a skilled operator and the cMo (CD14 ++CD16 -) percentages obtained by both analyses were compared. Information regarding age, gender, complete blood count, marrow cytomorphology, cytogenetics and molecular analysis was collected. Confirmed diagnoses were collected when available as well as follow-up for CMML patients. Results The comparison between cMo percentages from 267 PB files provided by the 10 centers and the centralized cMo percentages showed a good global significant correlation (r=0.88; p<0.0001; FigA) with no bias (FigB). Confirmed diagnoses were available for 212 files, namely 101 CMML according to the WHO criteria, 99 reactive monocytosis, and 12 MPN with monocytosis. A phenotype in favor of CMML, either classical with accumulation of cMo ≥94% or a bulbous aspect (FigC), was observed respectively in 81 and 14 patients. Hence, a total of 95 out of the 101 CMML patients translated into a sensitivity of 94% (FigD). Assessment of C reactive protein counts were available in seven of the 14 patients with the characteristic bulbous profile and correlated with an inflammatory state, showing a median of 93.0 [7.0-157.4] mg/L. Conversely, a phenotype not in favor of CMML (FigC) was observed in 83 of the 99 patients with reactive monocytosis and in 10 of 12 patients with MPN with monocytosis, leading to a 84% specificity (FigD). We established a Receiver Operator Curve (ROC) and again obtained a 94% cut-off value of cMo with an area under the ROC curve (AUC) of 0.865 (FigE). The second aim of this multicenter study was to assess the feasibility of the monocyte assay on 117 BM samples provided by 7 out of the 10 ELN centers, 43 of which being paired to PB samples. The comparison between cMo percentages provided by the 7 centers and the centralized cMo percentages showed a lower global significant correlation compared to PB samples (r=0.74; p<0.0001; FigF) with a slight underestimation of cMo percentage by the participating centers (FigG). The comparison between PB and BM samples cMo% obtained by centralized reanalysis showed an excellent global correlation (r=0.93; p<0.0001; FigH) with a higher percentage in the marrow (FigI). Seventy-nine files were associated to a confirmed diagnosis, as expected mostly CMML (n=69), only seven reactive monocytosis and three MPN with monocytosis. Thus, we determined a sensitivity of the "monocyte assay" on BM samples of 87% (a phenotype in favor of CMML being observed in 60 out of the 69 CMML with 6 bulbous aspect profiles) and a specificity of 80% (a phenotype not in favor of CMML being observed in 5 of the 7 patients with reactive monocytosis and in 3 of the 3 patients with MPN with monocytosis). Conclusions This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership.

Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies in myelodysplastic syndromes by theELN iMDSflow working group

“…In the case of CMML, a recent multi‐center effort by the ELN iMDS flow working group validated the ‘monocyte assay’ which complements the markers identified here (compare Table 8) and relies on the markers CD14 and CD16. An increase of ‘classical’ monocytes (CD14 ++ , CD16 − ) above 94% discriminates CMML from reactive monocytosis with high sensitivity and specificity (Wagner‐Ballon et al, 2023).…”

Multiparameter flow cytometry in the evaluation of myelodysplasia: Analytical issues

“…Talati et al (2017); Patnaik et al (2017); Hudson et al (2018); Tarfi et al (2018); Wagner‐Ballon et al (2023)…”

“…However, results of a survey concerning current MFC practice in 229 laboratories around the world showed that although many laboratories used large numbers of markers in MFC workup of MDS (median: 20 ± 4.5), the compliance with i MDSflow recommendations was low, and proposed scoring systems were not widely applied (Grille Montauban et al, 2019; Jensen et al, 2019). With the hope of increasing the harmonization of MFC MDS diagnostics, the current paper presents a summary of the progress in this field and an update on consensus i MDSFlow guidelines for the assessment of significant anomalies in various bone marrow (BM) cell compartments for MFC features of dysplasia as a part of a special Issue of Clinical Cytometry B, focused on MFC applications in MDS and MDS/MPN (Kern et al, 2022; van de Loosdrecht et al, 2023; van der Velden et al, 2023; Wagner‐Ballon et al, 2023; Westers et al, 2021; Westers et al, 2023).…”

Clinical application of flow cytometry in patients with unexplained cytopenia and suspected myelodysplastic syndrome: A report of the European LeukemiaNet International MDS‐Flow Cytometry Working Group

“…Using a cutoff value of >94% cMo monocytes by FCM CMML cases can be identified with a sensitivity of 92% and a specificity of 94%. In this Special Issue of Cytometry Part B, Clinical Cytometry, the ELN i MDS Flow WG shows the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity and also confirms the possibility of its use in BM samples (Wagner‐Ballon et al, 2021). Moreover, during successful therapy, the distribution of cMo, iMo, and ncMo reverts to a near normal or normal pattern.…”