Erythroid Lineage Analysis By Flow Cytometry Is of Highly Additive Value for MDS Diagnosis: A Study on Behalf of the HOVON89 Study Group
Authors: Cremers Eline MP , Westers Theresia M , Alhan Canan , Cali Claudia , Visser-Wisselaar Heleen A , Chitu Dana A , Stevens-Kroef Marian JPL , Silva Coelho Pedro , Vellenga Edo , Klein Saskia K. , Wijermans Pierre W. , De Greef Georgine E , Legdeur Marie-Cecile , Muus Petra , Ossenkoppele Gert J , Jansen Joop H , Van de Loosdrecht Arjan A 
Published: October 2019

Abstract
Flow cytometric analysis is a recommended tool in the diagnosis of MDS. It is used to underline the diagnosis based on dysplastic features by cytomorphology and typical cytogenetic abnormalities (gold standard). Current MDS flow cytometry (MDS-FC) scoring systems that evaluate the maturing myelo-/monocytic lineage reach a median sensitivity and specificity of approximately 71% and 93%. Since anemia is the most common feature in MDS, it was hypothesized that addition of in depth analysis of the erythroid lineage could increase the sensitivity of MDS-FC. We analyzed 176 bone marrow aspirates by MDS-FC, including 110 patients with MDS (low/int-1 risk) within a prospective multicenter clinical trial and 66 pathological controls (i.e. iron deficiency, iron incorporation disorders, vitamin B12 deficiency, cytopenia due to medication or chronic disease, aplastic anemia, and PNH). For erythroid lineage analysis, markers recommended by the ELNet iMDS-flow group including i.e. expression level of CD71 and CD36 and percentage of erythroid progenitors cells (CD117 positive or CD105 positive cells) were explored. Data from erythroid analysis were added to a previously described MDS-FC scoring system (Van de Loosdrecht JNCCN 2013). This scoring system combines the diagnostic MDS-FC score (Della Porta Haematologica 2012), analysis of myeloid progenitor cells, and of the granulocytic and monocytic cell compartment (Wells Blood 2003). At initial assessment the MDS-FC method categorized 75/110 MDS patients as ‘compatible with MDS’ (true positive), 23/110 as ‘minor MDS related aberrancies’, and 12/110 patients as ‘not compatible with MDS’ (false negative). Within the pathological control group: 45/66 were ‘not compatible with MDS’ (true negative), 20/66 showed ‘minor MDS related aberrancies’, and 1/66 ‘compatible with MDS’ (false positive). Calculated sensitivity and specificity of MDS-FC without taken into account erythroid analysis were 68% and 98%, respectively, in line with previous studies. Results of the erythroid FC analysis showed 82/110 MDS patients with clear dyserythropoiesis compared to 16/66 of the pathological controls. A strong correlation between presence of dyserythropoiesis by MDS-FC and the diagnosis of MDS as assessed by morphology was found (Pearson’s R=0.71; p<0.001). Results derived of the erythroid analysis were added to the MDS-FC scoring system. This resulted in: 94/110 MDS patients ‘compatible with MDS’, 13/110 patients with ‘minor MDS related aberrancies’, and 3/110 patients ‘not compatible with MDS’. Compared to 40/66 pathological controls that were ‘not compatible with MDS’, 23/66 that showed ‘minor MDS related aberrancies’, and 3/66 that were ‘compatible with MDS’. Although dyserythropoiesis by CM can be present in pathological controls, dyserythropoiesis by FC remained highly specific for MDS. Calculated sensitivity increased to 85%, with only a minor decline in specificity (95%). In conclusion, addition of the erythroid lineage analysis to a diagnostic MDS-FC score system led to an increased sensitivity, as validated within this prospective clinical trial. These data indicate that if translating to routine clinical practice, the erythroid integrated MDS-FC approach is highly instrumental in refinement of MDS diagnosis.

Disclosures
De Greef: Celgene: Consultancy. Van de Loosdrecht:celgene: Honoraria, Research Funding; alexion: Research Funding.