Evidence for Alternative Lengthening of Telomeres in Chronic Lymphocytic Leukemia Patients.

Telomere shortening is a consequence of repetitive clonal replication and leads to clonal deletion unless DNA extension and repair occur. All tumors must circumvent this problem by up-regulating mechanisms that lead to chromosomal lengthening. Two mechanisms have been identified that maintain chromosome ends- telomerase that does so by reverse transcription and alternative lengthening of telomeres (ALT) that occurs by homologous recombination. The latter function is characterized by the presence of promyelocytic leukemia protein-associated nuclear bodies (PML-NBs) and the presence of PML-NB is used to mark cells that use this process.

B cell Chronic lymphocytic leukemia (B-CLL) cells with unmutated Ig V genes have shorter mean telomere lengths compared with those exhibiting mutated Ig V genes. In addition, cells with unmutated Ig V genes demonstrate more telomerase activity than their mutated counterparts. The mutated cases show long and heterogeneously elongated telomeres in spite of the absence, in most cases, of detectable telomerase activity. Therefore we determined whether the ALT pathway plays a role in telomere maintenance in B-CLL, using a monoclonal anti-PML antibody and a flow-cytometric assay for assessment of PML protein. Telomerase-expressing Jurkat T cells and murine fibroblasts-L cells served as negative controls for PML staining, whereas the ALT positive Osteosarcoma cell line U2-OS served as a positive control. In a cohort of 20 B-CLL cases, PML protein was detected in all cases regardless of Ig V mutation status. In addition, a similar percentage of cells within the clones contained PML (10 - 90% of the members of unmutated clones and 11–96% of mutated clones), whereas peripheral blood B cells from 6/6 elderly normal donors did not show any PML staining. PML expression was compared with telomere length and telomerase activity in the same cases. The percentage of cells showing PML expression inversely correlated with telomerase activity (r= −0.58; p=0.029). Although in most published reports telomere maintenance by ALT occurs in the absence of telomerase activity, we found ALT (as suggested by PML positive cells) in cells with telomerase activity (detected by the standard TRAP assay). Thus, B-CLL cases can express PML bodies and some B-CLL cells can contain both PML-NB and express telomerase activity. These findings suggest that B-CLL cells can use two distinct mechanisms to assure telomere maintenance and perpetuate clonal survival and expansion.