Page 56 - 2022_03-Haematologica-web

M. Zapatka et al.
S5). In all patients, even in the ones with few methylation changes, hypermethylation was concentrated in poised promoters and polycomb-repressed regions, whereas hypomethylation occurred mostly in heterochromatin (data not shown; assignment of chromatin states was according to the published reference epigenome of CLL35). In spite of this common pattern, we could not identify any specific CpGs that consistently changed the methylation status throughout different patients or phases.
In summary, based on the patients analyzed here, the clonal evolution pattern seems to be linked to the disease phases, and increased changes in AF and a branched evo- lution are significantly more frequent after treatment com- pared to untreated patient phases (Figure 6).
Discussion
Medical history and disease course of patients with CLL is very individual. In this study, we examined WES data of CLL patients acquired at several time points during their disease and treatment course. Comparing consecutive samples from individual patients, we identified somatic mutations that were present in the leukemia cells and tracked over time the changes in AF of these mutations and the underlying fraction of cancer cells that carried the respective mutations. By modeling the clonal composition using the software TrAP,32 we discovered different clonal evolution patterns and disease progression courses that were linked to the treatment and response history of the patients (Figure 5). From the mutations and clonal changes that occur during CLL disease progression, we draw the following conclusions with respect to groups of genes, but also more conceptually with respect to clonal composition and evolution over time.
Recurrent mutations in genes were linked to CLL relapse in three different time- and treatment-dependent patterns.36 First, one subset of genes initially displays sub- clonal mutations that are enriched after therapy. In con- trast, mutations in a second set of genes remained clonally stable upon relapse. Finally, mutations in a third set of genes that are stable in most patients show clonal enrich- ment only in rare cases. However, these groups of muta- tions were not linked to a clinical phenotype. Furthermore, exponential-like growth patterns were recently associated with a larger number of CLL drivers and short time to first treatment.37 Of note, these data were derived from untreated CLL patients followed over time. In our patient cohort under the selective pressure of treatment, neither common genetic risk factors like IGHV or recurrent aberrations, nor variants or typically affected pathways are characteristic for a specific clinical course. And although the number of mutations increased slightly after treatment, this did not reflect or even predict out- come after therapy, nor did the number of (sub-)clones. Furthermore, clonal evolution was associated with treat- ment and indeed branched evolution was found more often in refractory cases, but not exclusively. These results reflect published data for relapsed cases after FCR thera- py, which could also not link progression-free survival to an evolution pattern after FC(R) therapy.4,22 Dividing our patient groups in long term responder and refractory cases allowed us in contrast to prior attempts to match the dura- tion of response to the extent of the clonal shift. Counterintuitively, clonal evolution that was mostly
dynamic and occurred primarily in patients who displayed refractory disease, i.e., where major changes in clonal evo- lution happened under the guise of a clinically stable or progressing disease. Therefore, what correlated most with the duration of response to treatment was a highly dynamic evolutionary change among sub-clones, and this change was directly associated with refractory disease. In contrast and unexpectedly, relapse after initially durable response occurred mostly with the same sub-clones. We identified three distinctly different courses of clonal evolu- tion that occurred under distinctly different treatment and response patterns. In refractory cases, clonal composition changed dramatically upon treatment failure and in patients 2, 4, and 18 this happened within only 3 months of therapy. Furthermore, in refractory phases, change in clonal composition was often accompanied by a profound shift in the bulk DNA methylation profile of the tumor, most probably reflecting different methylation profiles of the competing clones rather than de novo methylation changes, as it was previously shown that established CLL clones are epigenetically stable and changes in DNA methylation are unlikely to occur without genetic evolu- tion.24 As an example, patient HU-1-23 did not gain any new mutations between the two time points of his refrac- tory phase but underwent selection of particular pre-exis- tent CLL clones according to the branched genetic evolu- tion model and this was also manifested by a shift in DNA methylation of the bulk tumor. For the clinician managing the patient, “hidden“ selection of a resistant clone is masked by a tumor with a seemingly stable clinical phe- notype, i.e., with a persistent lymphadenopathy and leukocytosis. This dynamic clonal change suggests either
Figure 6. Model of the clonal composition changes. Model of the clonal composi- tion changes in the three different treatment phases (long-term untreated, relapsed and treatment refractory). Black lines indicate lymphocyte counts as sur- rogate marker for tumor load. Arrows indicate times of treatment. The stacked bar plot indicates clonal tumor composition where different colors indicate a different clone defined by a set of mutations.
612
haematologica | 2022; 107(3)