For several decades CLL has been defined as a chronic leukemia characterized by a passive accumulation of small neoplastic lymphocytes that do not proliferate and do not die. This definition has been revised in recent years as it was shown that CLL cells do proliferate mostly in proliferation centers. Because the increase in peripheral blood (PB) CLL cell count was lower than their proliferation rate, it was intuitively assumed that proliferation of CLL cells is accompanied by spontaneous apoptosis, and as proliferation rate increases so does the apoptosis rate. Because STAT3 is constitutively activated in CLL cells and provides CLL cells with survival advantage (Hazan-Halevy I. et al. Blood 115:2852, 2010), we wondered whether decreased levels of intracellular STAT3 would correlate with increased apoptosis of CLL cell.
Assessment of apoptosis rate by flow cytometry using propidium iodide (PI) and annexin V staining demonstrated that a significant fraction of freshly obtained PB CLL cells undergo spontaneous apoptosis in samples of 4 out of 4 patients with CLL. Spontaneous apoptosis was detected in 23% of CLL cells from a patient with a white blood cell count (WBC) of 16,000 (*10⋀6/L), in 20% of CLL cells from a patient with a WBC of 32,800, in 41 % of CLL cells from a patient with a WBC of 55,600, and in 65% of CLL cells from patient with a WBC of 101,000 (*10⋀6/L), suggesting that spontaneous apoptosis rates correlate with the number of circulating CLL cells. Because apoptotic cells are removed by the reticuloendothelial system, early apoptosis of CLL cells was assessed by quantification of cleaved PARP levels in CLL cells from 36 patients using an enzyme linked immunosorbent assay (ELISA). WBC in half of those patients ranged from 5,000 to 17,000 (*10⋀6/L) (median: 14,500) and in the other half from 151,000 to 680,000 (*10⋀6/L) (median: 237,000). The median level of cleaved PARP was three times higher in cells from patients with a high lymphocyte count than in cells from patients with a low lymphocyte count (P = 0.007), confirming our hypothesis that as disease burden increases so does CLL cell apoptosis rate. Because STAT3 plays a key role in CLL cell survival we sought to determine whether CLL cell apoptosis rates correlate with intracellular STAT3 levels. We quantified STAT3 levels in PB CLL cells from 185 CLL patients using an ELISA. Our data revealed a linear correlation between the number of CLL cells and intracellular STAT3 levels. The higher the lymphocyte counts, the lower were STAT3 levels (rp = 0.28, P<0.0001). Unlike WBC, STAT3 levels did not correlated with disease stage, cytogenetic abnormality, β2M levels, IgH mutation status, ZAP-70, or CD38. However, STAT3 levels were significantly higher in previously treated patients (No. = 24; P = 0.01), suggesting that previous exposure to chemotherapy activated compensatory survival pathways.
Spontaneous apoptosis occurs in PB CLL cells of all patients regardless of disease stage or cytogenetic abnormality. High PB lymphocyte count linearly correlates with increased spontaneous apoptosis rates and decreased intracellular STAT3 levels.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.