In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells

Abstract

Due to its relatively slow clinical progression, B cell chronic lymphocytic leukemia (B-CLL) is classically described as a disease of accumulation rather than proliferation. However, evidence for various forms of clonal evolution suggests that B-CLL clones may be more dynamic than previously assumed. We used a nonradioactive, stable isotopic labeling method to measure B-CLL cell kinetics in vivo. Nineteen patients drank an aliquot of deuterated water (2H2O) daily for 84 days, and 2H incorporation into the deoxyribose moiety of DNA of newly divided B-CLL cells was measured by gas chromatography/mass spectrometry, during and after the labeling period. Birth rates were calculated from the kinetic profiles. Death rates were defined as the difference between calculated birth and growth rates. These analyses demonstrated that the leukemic cells of each patient had definable and often substantial birth rates, varying from 0.1% to greater than 1.0% of the entire clone per day. Those patients with birth rates greater than 0.35% per day were much more likely to exhibit active or to develop progressive disease than those with lower birth rates Thus, B-CLL is not a static disease that results simply from accumulation of long-lived lymphocytes. Rather, it is a dynamic process composed also of cells that proliferate and die, often at appreciable levels. The extent to which this turnover occurs has not been previously appreciated. A correlation between birth rates and disease activity and progression appears to exist, which may help identify patients at risk for worsening disease in advance of clinical deterioration.

Figures

Figure 1

Modeling of the 2H2O levels in patients drinking 2H2O. (A) 2H2O enrichment data from the plasma of a CLL patient, showing that the dilution of 2H2O during the washout phase fits an exponential decay model. For the patient shown (CLL109), the equilibrium 2H2O enrichment was approximately 1.8% during the 2H2O intake period. Therefore, the daily water exchange rate, Wday, for this individual was approximately 2.3 l/d (2H2O intake of 60 ml/d × 70% 2H2O = 42 ml/d, divided by 0.018). The rate of the exponential decay is the fractional daily water exchange rate, fw, and, for the patient shown, was 0.065 per day. The total body water was therefore 35.4 liters (2.3/0.065). This individual weighed 55 kg, so the fraction of body weight due to water was 0.64. This fraction was used for all patients in the study to estimate total body water when a patient’s weight was available. When a patient’s weight was not recorded, an fw value of 6% per day was used. (B) Representative raw data for 2H2O enrichment in plasma and CD5+CD19+ cells from a B-CLL patient (CLL165). PMNL DNA saturates at a stable enrichment related to the 2H2O plasma enrichment. The vertical line at day 84 indicates the time at which the patient ceased intake of 2H2O. (C) Body 2H2O enrichment models for patients of various weights and fractional daily water exchange rates. Patients with high fw equilibrate faster, while patients with low fw can deviate substantially from a square pulse. (D) Body 2H2O enrichment model derived from a patient’s weight and equilibrium 2H2O enrichment accurately predicts the measured body 2H2O enrichment values.

Figure 2

Models for analysis of cellular 2H enrichment. (A) Single-compartment model in which 1 well-mixed pool is assumed. Unequal birth and death rates cause a change in pool size, but the labeled cell fraction is a function only of the birth rate (see Methods). (B) Two-compartment model in which cells proliferate in a distinct compartment (v1) and efflux into the sampled compartment (blood, v2). The birth, efflux, and death rates in all compartments were assumed to be equal. This model does not explicitly allow for the return of cells from the blood to the first compartment, but a high rate of exchange between the compartments relative to the birth rate would functionally equilibrate the model into a single compartment. Total clonal birth rate, B, is b / 1 + vr, where vr is the size of the second compartment relative to the first (v2/v1).

Figure 3

Labeling data, model fits, and WBC counts for the 19 B-CLL patients studied. Percent 2H enrichment in the DNA of leukemic cells was measured and converted into a fraction of labeled cells as described in Methods (squares, left axis). The single-compartment (dashed line) and 2-compartment (solid line) models were fit to the data using nonlinear curve fitting software. WBC counts taken during the study are shown (diamonds) plotted on a log scale at right. An exponential growth function was fit to the WBC data to provide a growth rate for the leukemic cells in the blood (Table 2).

Figure 4

Long-term follow-up samples are consistent with model assumptions. Samples from 4 patients were obtained more than a year after the end of the study. The labeled cell fractions were very close to the values predicted by the projected (dashed line) 2-compartment models that fit the earlier data (solid line).

Figure 5

Birth and death rates of B-CLL cells. The total clonal birth rates, B, are depicted in gray. The values were calculated from the 2-compartment model fitting parameters b and vr (see Methods) for those patients in which the r2 value of the model fit was greater than 0.6. The error bars represent SEs of the estimates. The growth rate trends are indicated across the top, as derived from Figure 3 and Table 2. The death rates were calculated by subtraction of the growth rate from the birth rate. The SE of the death rates was calculated from the square root of the sum of squares of the SE of the total clonal birth rates and the SE of the growth rate.