Biology Of

An observation made years ago is that CLL B cells isolated from the peripheral blood undergo rapid apopto-sis in vitro. The rate of apoptosis varies substantially, but there appears to be no simple correlation between rates of apoptosis in vitro and clinical outcome. A similar rapid rate of in vitro apoptosis is also observed in B cells derived from involved lymph nodes in patients with follicular lymphoma. As both populations express large amounts of BCL2 protein, these data may indicate that BCL2 alone is insufficient to suppress "spontaneous" apoptosis.

CLL cells in vivo do not appear to undergo such rapid apoptosis; work has been done to elucidate signaling pathways that maintain the viability in vitro, as definition of these pathways might define new therapeutic targets. A number of signaling pathways have been implicated, including IL4, IL7, CXCR4/SDF1, BAFF, and integrin signaling; the possible clinical relevance of these remains to be determined. Comparison of experiments is difficult because of differing culture conditions used. Serum-free conditions for the maintenance of CLL cells in vitro have been defined, but have not been widely used.43 Also, in most cases adequate definition of the CLL cells used in the experiments, in terms of both IGHV mutational and molecular cytoge-netic analysis, has not been performed.

Spontaneous apoptosis may be suppressed simply by either a) culturing at high density,44 most in vitro experiments use much lower concentrations of cells (106 cells/mL) than those actually seen in a patient, or b) culturing in the presence of albumin.45

Culturing CLL in the presence of adherent cells from a wide variety of sources, including marrow mesenchymal cells, may also prevent apoptosis.4647 The presence in the peripheral blood, specialized dendritic or "nurse" cells may maintain the viability of CLL for prolonged periods.1

Prevention of proteolytic degradation of the anti-apoptotic molecule MCL1 appears to be a key event in most of these experiments48; the rapid turnover of this protein may make it a target for a variety of therapeutic approaches. However, all of the above techniques do not result in proliferation of CLL cells, but rather in maintained viability. CLL cells in the blood are in the G0 phase of the cell cycle. As with spontaneous apop-tosis, a large number of simple manoeuvres, such as CpG dinucleotide stimulation along with IL2, or TNFa and IL6, can result in proliferation.49

However, the physiological and biological relevance of all of the above observations is not clear. Nor is it certain that cells derived from the peripheral blood are the correct population to be studied. Few studies have been done on either bone marrow or lymph node CLL cells, which may differ considerably from cells within the blood.

Moreover, a number of interesting studies in vivo suggest that CLL, rather than being a disease of suppressed apoptosis with only gradual accumulation of cells, may in fact, like other malignancies, be a disease of proliferation. First, there is evidence using metabolic labeling with D2O or heavy water and mass spec-trometry that patients with clinical stage A disease with stable peripheral blood lymphocytes may nevertheless turnover the entire clone in a matter of months 3. Secondly, and less directly, B-CLL cells have been shown to have significantly shorter telomeres than those in autologous neutrophils and B cells from healthy age-matched subjects; patients with unmu-tated IGHV genes had significantly shorter telomeres than those in mutated IGHV genes.50 51 These data suggest that a considerable number of cell divisions must have occurred in the leukemic cells after their genesis.

Taken together, these results indicate that CLL may be rapidly turning over in vivo and that several cell-cell interactions are required in order to maintain the viability of CLL cells. The necessity for persistent signaling suggests new therapeutic approaches.

There are also profound immunological defects in CLL whose biology remains poorly understood. Suppression of residual normal B cells as detected by low serum immunoglobulin levels is a feature, and worsens with disease progression. One explanation might be the release of immunosuppressive cytokines, such as IL10 or TGFp, from CLL B cells. However, there are also marked abnormalities of the T-cell populations in CLL. Somewhat surprisingly, their numbers are usually increased in CLL and their persistence may be essential for progression of CLL (reviewed in Ref. 16).

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