In Vitro Cell Division Analysis Reveals High Proliferative Potential and Clonogenicity within Primary Common-Type Human Acute Lymphoblastic Leukemia.
Authors: Nijmeijer Bart A. , Van Schie Marianke L.J. , Willemze Roelof , Falkenburg J.H. Frederik 
Published: April 2020

Abstract
Adult acute B-lineage lymphoblastic leukemia (ALL) has a poor prognosis. Recent studies suggest that primary ALL populations may be characterized by phenotypical and functional heterogeneity. Clonogenic ALL cells have been suggested to be a distinct subpopulation, with an extremely low frequency. Identification and elimination of these clonogenic cells is crucial for the development of novel therapeutic strategies. We have previously established a serum-free culture system for human ALL. The system has demonstrated to support long-term proliferation of clonogenic cells in 11 out of 23 investigated cases. The resulting LeidenALL-cell populations displayed the chromosomal abberations of the primary cells and engrafted into NOD/scid mice. Using this system, we studied the clonogenicity of primary ALL populations. Primary cells from three patients with common-ALL (CO, JV and MH) were labelled with CFSE. 16x106 cells were placed into culture and monitored for proliferation by FACS analysis of CFSE dye dilution. Six days after culture initiation proliferation of leukemic cells was evident, as 47%, 72% and 32% of parent (F0) cells had undergone 1 to 3 divisions in the CO, JV and MH cultures, respectively. After 12 days (CO and JV) or 33 days (MH) of culture, 100% of F0 cells had undergone mitosis demonstrating that all primary cells were capable of proliferation. At these time points the populations had undergone an average of 3.5, 4.5 and 3.4 divisions (range 2–5, 3–5 and 1–5 detectable divisions) in CO, JV and MH cultures, respectively. In contradiction to the occurrence of division in all cultures, absolute numbers of viable cells steadily decreased during 16 days after culture initiation, reaching a minimum of 2.5, 3.5, and 8.8x106 cells (16, 22 and 55% of input values) in the respective cultures. 16 days (CO and JV) and 30 days (MH) after culture initiation, viable cell numbers started to increase and followed log-phase kinetics with steady population doubling times of 3.0, 2.3 and 3.1 days, respectively. These observations suggest that during the first 3–5 divisions the cultures were characterized by the occurrence of proliferation as well as cell death. During the culturing period the cultures were periodically monitored for immune phenotype. All populations were homogenous throughout monitoring. The daughter cell populations displayed a stable CD10+CD19+CD20low immune phenotype similar to the immune phenotype of the F0 population. During culture a steady loss of CD34 expression and an increase of CD38 expression was observed. After 60 days of continuous proliferation the stable CD10+CD19+CD34lowCD38high phenotype characteristic of the long-term proliferating LeidenALL-cell populations was established. In conclusion, in contrast to previous studies, our assay reveals that in common-type ALL the primary population is capable of proliferation, contains a high level of clonogenicity, and therefore should be a target in novel therapeutic strategies.