Flow Cytometric Detection of the Ki-67 Proliferation Index and the Bcl-2 Anti-Apoptotic Index in Myelodysplastic Syndromes and Acute Myeloid Leukemia, and Their Clinical Implications
Authors: Mestrum Stefan G.C. , Vanblarcum Roanalis B.Y. , Drent Roosmarie J.M. , De Wit Norbert C.J. , Boonen Bert T. , van Hemert Wouter L.W. , Hopman Anton H.N. , Ramaekers Frans C.S. , Leers Mathie P.G. 
Published: November 2021

Abstract
Introduction: Standardization of the detection and quantification of leukocyte differentiation markers by the EuroFlow Consortium has led to a major step forward in the integration of flow cytometry in classification of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To further advance the integration and objectification of flow cytometry for characterization of these malignancies, more dynamic parameters assessing cell behavioral characteristics could prove useful, such as proliferative and (anti-)apoptotic markers. Proliferation and (anti-)apoptosis are processes that are tightly related to the pathogenesis, progression and chemo-/immunotherapy response of cancers. As a result, proliferation and (anti-)apoptotic markers have proven their value as objective parameters in the field of histopathology for diagnostic and prognostic applications in solid tumors and lymphoma. Although use of proliferative and (anti-)apoptotic markers as objective parameters in the diagnostic process of MDS and AML was studied in the past decades, this did not result in the incorporation of these biomarkers in their clinical diagnosis. The recent developments in flow cytometric analyses now allow the quantification of proliferative and (anti-)apoptotic fractions at the level of individual maturing bone marrow cells. Therefore, we aim to determine the Ki-67 proliferation indices and Bcl-2 anti-apoptotic indices in maturing bone marrow cells in order to assess whether these parameters could have future clinical implications for the diagnostic work-up of MDS and AML.
Methods: Fifty bone marrow aspirates from femoral heads of non-malignant cases, 20 aspirates of MDS patients and 20 aspirates of AML were included in this study. Ten-color flow cytometry in combination with a software-based maturation tool was used for analysis of the Ki-67 proliferative and Bcl-2 anti-apoptotic indices of blast cells and during the erythro-, myelo-, and monopoiesis.
Results: Ki-67 proliferative indices of blast cells and immature erythroid, myeloid and monocytic cells were significantly lower in MDS patients compared to the non-malignant cases, while the Bcl-2 anti-apoptotic indices were significantly elevated in these cells. Furthermore, the Bcl-2 anti-apoptotic indices were also increased in mature erythroid, myeloid and monocytic cells of MDS patients. The decreased Ki-67 proliferative indices and increased Bcl-2 anti-apoptotic indices in blast cells and erythroid, myeloid and monocytic cells were even more prominently observed in AML patients.
Conclusions: The lowered Ki-67 proliferative indices and elevated Bcl-2 anti-apoptotic indices in blast cells and immature progenitor cells led to a better understanding of the pathophysiology of MDS and AML, and explained the low chemotherapy response of these patients. Side-effects of such therapies can also be explained by the Ki-67 proliferation indices and Bcl-2 anti-apoptotic indices. Moreover, the increase of the Bcl-2 anti-apoptotic fraction is an important factor in the progression of MDS to AML. Future studies on the clinical applications of these parameters for MDS and AML are necessary and can include many applications, such as prediction of chemo-/immunotherapy response, diagnostic and prognostic applications.

Disclosures
Ramaekers: Nordic-MUbio: Current Employment.