Increased Apoptosis of Peripheral Blood and Bone Marrow B and T Cells Correlates with Advanced Stages and Poor Risk Factors in Patients with B-CLL
Authors: Sieklucka Malgorzata , Bojarska-Junak Agnieszka , Surdacka Agata , Hus Iwona , Wasik-Szczepanek Ewa , Dmoszynska Anna , Wach Malgorzata , Lewandowska Magdalena , Rolinski Jacek M. 
Published: April 2020

Abstract
B-cell chronic lymphocytic leukemia (B-CLL), is characterized by the accumulation of long-lived, neoplastic B-lymphocytes in peripheral blood, bone marrow and secondary lymphoid organs. Apoptotic processes have been shown to be altered in leukemic B cells, however, the role of apoptosis in the mechanisms of disease progression remains unclear. Recent studies suggest that the clonal excess of B-cells is caused not only by a decrease in cell death but also by increased cell proliferation. We have recently reported on a high rate of apoptosis leukemic B cells in peripheral blood (PB) of advanced stage patients and that apoptosis of PB lymphocytes from advanced-stage (III–IV acc. Rai) patients is higher than that in early-stage (0–II acc. Rai) patients. However the spontaneous apoptosis in B-CLL patients was significantly lower compared to the healthy controls that confirmed the defective apoptosis as one of the mechanisms of leukemic lymphocytes accumulation in B-CLL. Continuing our research, in the presented study we measured apoptosis of B and T cells in peripheral blood and bone marrow in correlation with the stage of B-CLL and prognostic factors. Materials and methods: Peripheral blood and bone marrow (BM) samples were obtained from 120 previously untreated B-CLL patients. An analysis of apoptosis within the B and T cells population was performed using flow cytometer and chloromethyl-X-rosamine staining (Mito Tracker Red CMXRos). CMXRos was used to detect disruptions in the mitochondrial membrane potential (ΔΨm), which is one of the earliest events in the apoptotic pathway and allow finding apoptotic cells when there are still in PB and BM. We found that ex vivo lymphocyte apoptosis was higher in BM compared to PB (p&lt;0.05). Moreover, both B-cell and T-cell apoptosis in BM was higher than in PB (p&lt;0.0001 and p&lt;0.001, respectively). When compared, ex vivo apoptosis of T cells was found higher than that of B cells, both in BM (p&lt;0.0001) and PB (p&lt;0.0001). The percentage of apoptotic leukemic B cells correlated negatively with Bcl-2/Bax ratio in CD19+ B cells (p&lt;0.05). Similarly, the percentage of apoptotic CD3+ cells correlated negatively with Bcl-2/Bax ratio in CD3+ cells (p&lt;0.01). We also found that the percentage of apoptotic leukemic B cells correlated positively with the expression of proapoptotic protein Par-4 (prostate apoptosis response-4) in CD19+ B cells (p&lt;0.01). The expression of Par-4 protein in CD19+ B cells correlated positively with the percentage of CD38+ cells (p&lt;0.05), and it was higher in patients with CD38+ and ZAP-70+/CD38+ phenotypes (p&lt;0.05 and p&lt;0.01, respectively). There was a positive correlation between the expression of Par-4 protein and the lactate dehydrogenase (LDH) and β2-microglobulin serum concentrations (p&lt;0.01 and p&lt;0.05, respectively). Furthermore, the percentage of apoptotic CD19+ cells correlated positively with the LDH serum level (p&lt;0.05). These data indicate that high amount of apoptotic leukemic cells in PB and BM might be considered as poor prognosis factor. Higher rate of B and T cells apoptosis in BM than in PB suggest the influence of bone marrow microenviroment on this process. Our results indicate also that high rate of T cells apoptosis might be responsible for immune dysfunction including both impaired anti-infection immunity as well as impaired anti-cancer response resulting in disease progression.