Clonal Evolution in Chronic Lymphocytic Leukemia

The phenotypic characterization of dominant CLL clones at various disease stages using multiple experimental approaches has deepened our understanding of CLL biology and clinical behavior. However, much less is known about the phenotype and phenotypic changes that occur in CLL over time and with disease progression when measured longitudinally. Analysis of large CLL cohorts at various disease time points is needed to better capture the dynamics of this disease.

A total of 143 CLL cases were surveyed at trial enrollment and longitudinally for changes in acquired genomic copy number aberrations (aCNA), acquired uniparental disomy (aUPD), and somatically acquired mutations in the genes TP53, SF3B1, and NOTCH1. On an unselected basis, longitudinal samples were studied at a minimum of 24 months after initial sample procurement in the case of no therapeutic intervention or at the time of relapse after chemotherapy. The median time span between enrollment and longitudinal samples was 40.5 months for samples without intervening therapy (range 9–79 mos.) and 42 months for relapsed cases (range 7–74 mos.). One hundred eight cases did not receive therapeutic interventions in-between sample procurements, while 35 were relapsed from therapy. In 13 cases, time spans less than 24 months were allowed if the patient carried a high-risk CLL genomic feature or if limited follow-up samples were available. Cells were purified by flow cytometry sorting of viable CD19+ and CD3+ cells. DNA samples isolated from paired tumor and normal samples were studied for aCNA using Affymetrix SNP 6.0 arrays and the software program dChip. Samples were also analyzed for LOH/aUPD via internally-developed software. aCNA were scored through visual interpretations of the dChip display of paired samples and corroborated through an algorithmic lesion calling method. Finally, the genes TP53 exons 2–10, SF3B1 exons 13–17, and NOTCH1 exons 34 were resequenced in all samples.

The dominant phenomenon observed in this large CLL cohort was genomic stability for aCNA and gene mutations, which was detected in 116 (81%) of cases analyzed. Importantly however, 27 (19%) of CLL cases differed between enrollment and longitudinal follow-up. Within this subgroup of cases, 21 cases harbored changes in aCNA/LOH/aUPD, while 7 cases carried a change in the mutation status of TP53, NOTCH1 or SF3B1 (one case had both aCNA and mutation changes). Specifically, 19 cases with aCNA/LOH/aUPD changes added lesions at longitudinal analysis and lost none: clear examples of clonal evolution. Four cases added TP53 mutations and two cases added a NOTCH1 mutation. Conversely, TP53 mutations were never lost, while one case with a novel TP53 mutation lost a pre-existing NOTCH1 mutation and one case lost a SF3B1 mutation. No case gained a SF3B1 mutation. Only one CLL case at relapse demonstrated an aCNA/LOH/aUPD pattern that was completely unrelated to the dominant CLL clone at enrollment.

In this report, we describe genomic changes in a cohort of 143 CLL cases analyzed longitudinally. This study constitutes the largest analysis of such changes reported to date. Our overall estimates of genomic instability in CLL are likely underestimates of the true frequency, given the observed natural selection against aggressive CLL inherent in this type of analysis. From this data several conclusions can be reached that have implications for our understanding of CLL clonal dynamics and the effects of therapy on clonal evolution/emergence: i) the vast majority of genomic changes are acquisitions of new lesions/mutations combined with the retention of pre-existing lesions–therefore persistence of antecedent clones underlies CLL relapse and disease persistence; ii) CLL cases demonstrating clonal evolution were enriched for cases that received intervening chemotherapy (∼50% in this cohort); iii) the acquisition of TP53 mutations followed intervening chemotherapy, providing further impetus to carefully select patients for chemotherapy; iv) NOTCH1 mutations were rarely acquired over time and in one case lost; v) no SF3B1 mutations were acquired over time and in one case lost; vi) the latter two observations argue against a strong driver role for these mutations in CLL progression/relapse; vii) the acquisition of aCNA/LOH/aUPD and gene mutation was non-overlapping, indicative of distinct roles for each aberration type in CLL evolution.

No relevant conflicts of interest to declare.