The Landscape of Dynamic Genetic Changes in Ibrutinib-Treated CLL

Cancer evolution poses a significant challenge to the ability of therapy to eliminate malignant cells. In chronic lymphocytic leukemia (CLL), clonal evolution was reported as a key feature of disease relapse after standard chemoimmunotherapy and after targeted therapy with the BTK inhibitor ibrutinib. To study potential selection of pre-existing sub-clones, we analyzed changes in clonal architecture at early time points in patients receiving ibrutinib therapy. We therefore performed in-depth longitudinal analysis of CLL samples collected from 59 patients treated with ibrutinib alone (n=44) or in combination with rituximab (n=15).

Across this cohort, we performed whole-exome sequencing (WES) on a median of 3 samples per patient with median coverage of 105x (range 50-225x). Pretreatment samples were available for all patients, with post-treatment samples at 1 and 6 months for more than 85% of patients. Additional samples were available at 2, 3, or 12 months for 22 patients. The median age of the cohort was 65 (range 33-85) years. Thirty-one patients (53%) had del(17p) by FISH, 37 patients (63%) had IGHV unmutated CLL and 32 patients (54%) had relapsed or refractory disease. We observed a median of 56 (range 17-201) somatic silent and non-silent single nucleotide variants (SNVs) and insertions and deletions (Indels) per patient. The number of SNVs and indels did not differ between patients receiving ibrutinib alone or with rituximab (Wilcoxon, P=0.2). Thirteen patients had somatic mutations of BCR pathway genes prior to treatment, including one with CARD11-L251P, previously reported to confer resistance to ibrutinib in diffuse large B-cell lymphoma.

Comparing pre-treatment and the latest samples within the first year (range 56-357 days, median 168), 26 of 59 (44%) CLLs showed significant changes in cancer cell fraction (CCF) over time, defined as a shift > 5% CCF of the clone with the greatest change (BH-FDR <0.1). These included 11 CLLs with a clone that significantly increased in size, 9 with a clone that significantly decreased in size, and 6 with significant reciprocal rises and falls of different clones consistent with a branched evolutionary pattern. The median CCF change during the observation period was 24% (range 7-89%). Thus, even within a short period of treatment exposure, dense temporal sampling of CLLs reveals that ibrutinib therapy commonly leads to clonal shifts.

The likelihood for clonal shifts was not impacted by whether patients were treated with ibrutinib alone or with rituximab (45% vs. 40%, P=0.77), nor by exposure to prior therapy (47% vs. 41% with and without prior therapy, P=0.79). We observed trends towards higher likelihood of early clonal evolution in CLLs with unmutated IGHV status, with TP53 somatic aberrations, and those with a pre-treatment subclonal driver (subclones containing CHD2, BIRC3, SF3B1, MAP2K1, CARD11, XPO1, TP53, PTPN11, KRAS, HIST1H1E, ATM). Finally, patients with a decrease in ALC at 6 months compared to pre-treatment were more likely to exhibit significant CCF shifts (P=0.01). This last observation suggests that effective treatment activity and resulting therapeutic bottlenecks indeed contribute to the shift in clonal proportion within the first year of ibrutinib therapy.

Relapse samples were available for 10 CLL cases, 6 of whom showed early clonal shifts during the first year of therapy. These included 2 cases of transformation to prolymphocytic leukemia (PLL), 2 with Richter transformation (RT) and 6 cases of progressive CLL disease. All relapse cases were clonally related to the pre-treatment samples. Mutations in BTK or PLCg2 were found in 5 cases by either WES or deep targeted sequencing of known resistance hotspots (both PLL, one RT, two progressive CLL). The remaining 5 cases showed marked clonal shifts suggestive of selection of other resistant genotypes.

Altogether, targeted therapy with ibrutinib is associated with overall clonal stability in more than half of CLLs over the first year of treatment. Nevertheless, in many patients, clonal selection occurs even during the first months of therapy and may provide clues regarding the future evolutionary and clinical outcome of the disease.