Correlation of Leukemia-Cell Birth Rate Measured by Heavy Water Labeling with Other Prognostic Markers in Early Stage Chronic Lymphocytic Leukemia.

Abstract 60

Chronic lymphocytic leukemia (CLL) was formerly considered an accumulative disease of immunologically incompetent lymphocytes that have a slow rate of cell turnover. However, recent studies suggest that CLL cells of some patients may have an unexpectedly high birth rate, which in some subjects, is counterbalanced by a relatively high death rate. In particular, studies on the leukemia cells of patients who ingested heavy water (2H2O) revealed that some patients had leukemia cells that incorporated deuterium into DNA at rates consistent with a birth rate of 1% of the leukemia population per day. This high birth rate, when counterbalanced with comparable rates of leukemia cell turnover, allows patients to maintain a steady state that was suggestive of indolent disease. Preliminary data also suggested a relationship between disease activity and CLL cell birth. To examine the association between measured rates of leukemia-cell turnover with established surrogate markers of aggressive disease, the CLL Research Consortium (CRC) recently completed enrolment of 119 patients with early stage disease. Patients were to be untreated, Rai Stage 0, 1, or 2 and diagnosed within the previous 3 years. The leukemia cells of each of these patients were evaluated by the central tissue core of the CRC for expression of ZAP-70, CD38, and immunoglobulin heavy chain variable region gene (IGHV) mutation status. We report here a subset analysis of baseline data for 59 patients (57% male, age 58.7 ± 10.1 y). Thirty-seven percent of patients (n=22) had leukemic cells that expressed unmutated IGHV, 39% (n=23) had cells that expressed ZAP-70 (> 20% positive by cytoplasmic flow cytometery), and 19% (n=11) had CD38 positive cells (> 30% positive by multiparameter flow cytometry). Associations between the traditional prognostic markers were exceedingly strong (ZAP-70 with mutation status, p < 0.001; ZAP-70 with CD38, p = 0.013; CD38 with mutation status, p =< 0.001). Birth rate was expressed as the peak percent new cells per day calculated from 2H enrichment in DNA using isotope ratio mass spectrometry (IRMS). Data from the previous study suggested more aggressive disease activity associated with a birth rate above 0.35% per day. Birth rates in the present study ranged from 0.07 - 1.31 % per day, spanning both sides of the previously published 0.35% per day cut-off. Using the Wilcoxon rank sum test, patients whose clones expressed unmutated IGHV, were ZAP-70 positive, or were CD38 positive had a significantly higher daily leukemia cell birth rates than subjects who were IGHV mutated (median of 0.39 v 0.24, p = 0.01), ZAP-70 negative (median of 0.36 v. 0.22, p = 0.002), or CD38 negative (0.62 V. 0.24, p = 0.001). Thus, we have shown that CLL cell birth rates can be determined in a multi-centered trial in a large number of patients, and that patients can be stratified into groups with varying rates of perceived cell turnover. The cell turnover data correlate directly in multivariate analysis with established surrogate markers of aggressive disease, a poor clinical course and decreased survival with higher birth rates associating with unmutated IGHV and expression of ZAP-70 and CD38. Clinical follow-up of these patients is underway to determine if these four parameters serve as complementary prognostic markers. Nevertheless, the strong association between established surrogate markers of aggressive disease with high leukemia-cell birth rates suggests that high-rates of leukemia-cell turnover define a biology that is associated with adverse outcome. As such, these studies strengthen the notion that leukemia cell populations that have high rates of cell turnover can more rapidly accumulate secondary and tertiary genetic mutations that potentially govern the progression of this disease.