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Clonal evolution in relapsed/refractory CLL cases
an increased evolutionary capacity in these patients or simply the presence of at least one resilient clone. Importantly a selective pressure of therapy is necessary to induce or catalyze this clonal change as provided by the clinical course of patients 15 and 19. Both have a long term untreated phase without marked evolution but a strong shift after becoming refractory. Therefore, neither the underlying risk factors, the natural disease biology nor the type of evolution, which is branched in untreated and refractory phase in both patients, reflects the clinical course while the extent of evolution does. Importantly, we could show in addition that this process occurs also independently of TP53 mutations, i.e., in a cohort of CLL patients without TP53 aberrations before treatment. Thus, in these patients the clonal evolution is driven mostly by treatment that seems to select for resistant CLL clones.
Thus, our key finding is the striking observation of a clonal turnover during therapy in those patients, who were considered treatment refractory and therefore are assumed to have a stable tumor load.
In contrast, long term untreated cases and late relapses are genomically stable, although they are observed over a much longer period of time. In the latter we found a remarkably stable genomic landscape considering that these patients received a therapy with a subsequent re- growing after a prolonged treatment-free interval. This stability is completely different to a tumor that is refrac- tory and apparently unaffected by therapy, but in con- trast displays a dramatic change in clonal composition. This opposing clinical and genomic phenotype at first appears counterintuitive. However, these different cours- es of clonal dynamics in relapsing and refractory patient phases could be explained by the preexistence of a resist- ant clone that after removal of the bulk tumor by a treat- ment intervention will quickly grow out and fill the empty niche. If such resistant clones are absent, compe- tition and outgrowth over time is still possible, so that the tumor regrows with an almost identical clonal com- position. This finding mechanistically explains and underlines the relevance of the widely used clinical para- digm of repeating the previous treatment regimen when a good and long-lasting response is achieved: based on the same clonal composition at relapse, the clinician can expect another good response of the tumor to the treat- ment as the tumor has the same clonal composition as before the treatment. Interestingly, new treatment modalities like venetoclax may behave similarly due to the strong reduction of the tumor load, comparable to chemotherapy: while patients treated with short and effective venetoclax containing combination therapies lack BCL2 mutations at relapse, refractoriness to a long lasting venetoclax treatment associates with the out- growth of a BCL2 mutated clone displaying the same clonal shift towards drug resistance, that we observe here in chemotherapy refractory cases.38,39 On the other hand, ibrutinib may cause a more decelerated clonal shift due to its slow debulking treatment effect and also only slowly emerging resistant clones (i.e., point mutations in BTK/PLCG240).
In summary despite the small patient cohort (n=25 with 54 time points), we identified a link between changes in the variant AF and changes in clonal architecture, both of which are linked with shortened time to further treat- ment, i.e., treatment resistance. We found clonal evolu- tion to occur without strong contribution of known CLL driver genes. However, there is a dramatic difference in clonal evolution patterns between relapsed and refractory samples, which highlights the importance of the treat- ment-induced clonal changes in relation to treatment response. This intrinsic characteristic of CLL evolution underlines the relevance of comparing the benefits of treatment compared to the watch-and-wait strategy that has a very low clonal evolution rate. Furthermore, the substantial clonal evolution in refractory disease high- lights the need for novel, non-genotoxic treatment regi- mens with targeted therapy that are less likely to induce clinical disease resistance by selecting out preexistent refractory sub-clones.
Disclosures and Funding
DM was supported by DFG (SFB1074 subproject B1 and B2) and ERA-NET “FIRE-CLL”; MZ and MS were supported by ERA-NET “FIRE-CLL” and BMBF “PRECISE”; SS received support from DFG (SFB1074 subproject B1 and B2, BMBF “PRECISE” and ERA-NET “FIRE CLL”, he also received honoraria and research support from AbbVie, AstraZeneca, Celgene, Gilead, GSK, Hoffmann La-Roche, Janssen, Novartis; ET received honoraria from the Speakers Bureau, Advisory board and travel support of Hoffmann La- Roche and AbbVie.
Contributions
PL and SS developed concepts and ideas; MZ developed soft- ware; ET applied methodology; ET validated data; MZ, ET and DYY performed formal analysis; MZ, ET and SÖ performed investigations; TZ, CS, JB, HD, ET, PL and SS provided resources; MZ, ET, and SS cured data; MZ, ET, SÖ, MS, DM, PL and SS wrote the original draft; MZ, ET, SÖ, DYY, MS, TZ, CS, JB, DM, PL and SS wrote, reviewed and edited the manuscript; MZ, ET, SÖ, DYY and DM visualized concepts and data; PL and SS supervised the project; PL and SS were in charge of project administration; MZ, ET, TZ, PL and SS acquired funding.
Acknowledgements
We thank all patients and physicians, especially Andrea Schnaiter, for donating samples and participating in this study. We thank Michael Hain and Rolf Kabbe for computational sup- port. We thank Stephan Wolf and the High Throughput Sequencing unit of the Genomics & Proteomics Core Facility, German Cancer Research Center (DKFZ), for providing excel- lent sequencing services.
Data availability
Sequencing data have been deposited at the European Genome-Phenome Archive hosted at the EBI under accession EGAS00001003652. Data for the methylation arrays are acces- sible at GSE143411.
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