Expression of CD38 and High Levels of CD5 Identifies Members of CLL Clones That Have Progressed beyond G1 and Proliferated.

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the monoclonal expansion of CD5+ B cells with the morphology of small mature lymphocytes. Although the great majority of circulating CLL B cells appears as quiescent/non-proliferating lymphocytes, a small percentage of cells in peripheral blood appear to be cycling. Furthermore, within these clones, the CD38+ subset is enriched in those cells that entered G1, as evidenced by a larger number of Ki67-expressing cells. In an attempt to further identify submembers of CLL clones with different proliferative capacities, we measured, by flow cytometry, expression of Ki67 along with mcm6 and Cyclin D1, expressed from G1 to M, and Cyclin B1, expressed in G2-M. Since the expression of CD5 is characteristic of CLL cells and increases in vitro upon cellular activation, the intensity of CD5 expression was used to further divide CD38+ and CD38− cells into CD5bright and CD5dim fractions. The leukemic clones of eighteen IgVH unmutated CLL (U-CLL) and 16 IgVH mutated CLL (M-CLL) patients were analyzed. A similar pattern of expression for each cell cycle related marker studied was found. The presence of CD38 and high expression of CD5 was correlated with increased percent of cells expressing the various cell cycle markers (CD38−CD5dim<CD38−CD5bright; CD38+CD5dim<CD38+CD5bright). Strikingly, significant differences characterized the intraclonal comparisons between the 4 subpopulations (Table). To confirm these findings, the cells of 4 CLL patients involved in a deuterated “heavy” water protocol were sorted using CD38 and CD5 expression. In two of these cases, because of limiting cell numbers, CD5bright and CD5dim cells could be obtained only among CD38− cells. CD38−CD5bright cells contained more deuterium in their DNA than CD38−CD5dim cells indicating that more CD38−CD5bright cells had divided than CD38−CD5dim cells. A similar pattern was found in two patients for whom all four subfractions could be sorted. Thus, lowest deuterium enrichment in DNA was found in CD38−CD5dim cells and highest in CD38+CD5bright cells. The combined use of surface and intracellular markers, together with the 2H labeling, effectively further subdivides the proliferative leukemic compartment in CLL. Expression of Cell Cycle related markers in CLL B cells defined by surface expression of CD38 and CD5 N=34 mcm6 % CyclinD1 % Ki67 % CyclinB1 % For every marker studied: all intraclonal comparisons (1 vs. 2, 1 vs. 3 etc.) are statistically significant (P<0.05) with the exception of 2 vs. 3 and, for cyclinB1, 3 vs. 4 CD38−CD5dim (1) 4.0±0.8 9.9±2.6 3.8±0.8 0.8±0.2 CD38−CD5bright (2) 9.8±1.9 17±4.2 10.2±1.9 2.1±0.5 CD38+CD5dim (3) 8.5±1.4 13.7±2.8 7.8±1.1 2.3±0.6 CD38+CD5bright (4) 19.5±2.3 24.3±4.0 15.2±1.8 2.8±0.6