A subset of blood mononuclear cells from patients with B-cell chronic lymphocytic leukemia (CLL) can differentiate into large, adherent cells, termed “nurse-like cells” (NLC), that can attract CLL cells and protect them from undergoing apoptosis in vitro. When removed from NLC in vitro, CLL cells often undergo spontaneous apoptosis. We observed that the relative viability of isolated CLL cells cultured without versus with NLC appeared constant for any one sample in different experiments and did not vary with the source of the NLC used in any one experiment. Furthermore, the isolated CLL cells from some patients could survive in vitro without NLC better than could the leukemia cells of other patients. We examined for characteristics associated with CLL cells that were more or less reliant on NLC for survival in vitro. For this, we defined CLL cell populations as having “low dependency” on NLC when the relative leukemia cell viability after two days culture without NLC was less than one half-log (33%) lower than that of the same CLL cells co-cultured with NLC. Using a Multiplex Ligation-dependent Probe Amplification (MLPA) assay, we examined CLL cell populations with low dependency (n = 4) versus samples with high dependency (n = 5) for expression of 40 distinct mRNA that encoded known pro-apoptotic or anti-apoptotic proteins. CLL cells with low dependency expressed significantly higher levels of BCL-2, and significantly lower levels of BAX-2 than CLL cells with high NLC dependency. There was no significant difference between the mean white blood cell counts of patients whose CLL cells had low-dependency versus those whose cells had high dependency on NLC in vitro. However, in the high dependency group there was a significantly higher proportion of cases that expressed ZAP-70 than in the group with low dependency. This association, however, could reflect the fact that many of the samples with low dependency were acquired from patients who had long-established disease, a characteristic that typically was not associated with patients who have CLL cells that expressed ZAP-70. The mean time from diagnosis to the date of sample collection was significantly longer (m = 66.6 ± 47.0 months, median = 49 months, n=30) for samples with low dependency than it was for those cases with high dependency (m = 41.2 ± 25.1 months, median = 39.5 months, n=20) (p=0.017). Furthermore, evaluation of serial samples collected over years from the same patients (n = 5) revealed that the relative dependency of CLL cells on NLC could decrease significantly over time. In none of the serial samples did we observe increased dependency of the CLL cells on NLC over time or over that observed for the CLL cells isolated from the initial sample collection. These data suggest that over the course of the disease CLL cells might acquire less dependency on NLC, and potentially also on its stromal microenvironment.

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