Clonal evolution leading to ibrutinib resistance in chronic lymphocytic leukemia

Abstract

Disease progression in patients with chronic lymphocytic leukemia (CLL) treated with ibrutinib has been attributed to histologic transformation or acquired mutations in BTK and PLCG2. The rate of resistance and clonal composition of PD are incompletely characterized. We report on CLL patients treated with single-agent ibrutinib on an investigator-initiated phase 2 trial. With median follow-up of 34 months, 15 of 84 evaluable patients (17.9%) progressed. Relapsed/refractory disease at study entry, TP53 aberration, advanced Rai stage, and high β-2 microglobulin were independently associated with inferior progression-free survival (P < .05 for all tests). Histologic transformation occurred in 5 patients (6.0%) and was limited to the first 15 months on ibrutinib. In contrast, progression due to CLL in 10 patients (11.9%) occurred later, diagnosed at a median 38 months on study. At progression, mutations in BTK (Cys481) and/or PLCG2 (within the autoinhibitory domain) were found in 9 patients (10.7%), in 8 of 10 patients with progressive CLL, and in 1 patient with prolymphocytic transformation. Applying high-sensitivity testing (detection limit ∼1 in 1000 cells) to stored samples, we detected mutations up to 15 months before manifestation of clinical progression (range, 2.9-15.4 months). In 5 patients (6.0%), multiple subclones carrying different mutations arose independently, leading to subclonal heterogeneity of resistant disease. For a seamless transition to alternative targeted agents, patients progressing with CLL were continued on ibrutinib for up to 3 months, with 19.8 months median survival from the time of progression. This trial was registered at www.clinicaltrials.gov as #NCT01500733.

Figure 1.

PFS. Kaplan-Meier estimates of PFS of (A) all patients and subgroups stratified by: (B) TP53 aberration (absent vs present), (C) prior treatment (RR CLL vs TN), (D) Rai stage (I/II vs III/IV), (E) B2M (≤4 mg/L vs >4 mg/L), and (F) IGHV mutational status (mutated IGHV [M] vs unmutated IGHV [U]). Among patients with TP53 aberration (n = 53), subgroups are further divided by: (G) prior treatment (RR vs TN), (H) Rai stage (I/II vs III/IV), and (I) B2M (≤4 mg/L vs >4 mg/L).

Figure 2.

BTK and PLCG2 mutations at disease progression. Schematic representation of functional domains of (A) BTK and (B) PLCG2 with amino acid substitutions due to nonsynonymous mutations or deletion indicated. (A) BTK gene domains and nucleotide changes. Mutations in exon 15 of BTK affect C481 in the protein tyrosine kinase (PK) domain. BTK c.1442G>C (red circle and red triangle) and c.1441T>A mutations lead to C481S. BTK c.1441T>C mutation leads to C481R substitution. (B) PLCG2 gene domains and nucleotide changes. Mutations in PLCG2 exons 19 and 20 affect the N-terminal SH2 domain and mutations in exon 24 the pleckstrin homology (PH) domain. The P664S mutation and the 6-nucleotide deletion in PLCG2 exon 20 (*), have not been previously described in CLL. (C) Number of patients with BTK and/or PLCG2 mutations at progression. (D) Sanger sequencing and NGS of patient PD9 reveals 3 different types of nucleotide changes in BTK exon 15. Shown is the sense DNA strand. BTK is encoded on the antisense strand; the black arrow indicates the read direction. Dotted and solid lines are aligned at BTK nucleotide position 1441. Left panel, The result of Sanger sequencing showing c.1442G>T (C481S), c.1441T>C (C481R), and c.1441T>A (C481S). Right panel, The result of NGS showing c.1441T>C (C481R) and c.1441T>A (C481S). EF, EF-hand domain; MUT, mutated; PI, phosphatidylinositol-specific phospholipase C X domain; WT, wild-type.

Figure 3.

Clonal evolution during treatment with ibrutinib. (A) A swimmer plot of 15 patients who progressed on ibrutinib. Each lane represents an individual patient. Numbers at the end of the line indicate TTP (months). Open diamonds indicate BTK and PLCG2 candidate regions were tested for mutations, and no mutation was detected. Red triangles indicate the detection of BTK mutations. Blue triangles indicate PLCG2 mutations. (B) Growth kinetics of mutant clones. Three representative cases with BTK and/or PLCG2 mutations are shown (PD8, PD9, and PD11). Red color indicates BTK mutation. Blue color indicates PLCG2 mutation. The cell count of mutant clones was estimated from the VAF assessed by ddPCR (supplemental Table 2), the ALC, and the proportion of B cells (CD19⁺). Time points with VAFs below the lowest limit of detection (0.2%) are plotted as 1 cell (10°).

Figure 4.

Biomarkers of disease progression. ALC in panel A and serum-derived biomarkers were measured at baseline (Pre), best response (Best), and progression (PD) in matched samples: (B) B2M; (C) LDH; (D) clonal light chain (LC), either κ or λ, corresponding to the light chain restriction of the CLL cells as determined by flow cytometry; and BCR-regulated chemokines (E) CCL3; and (F) CCL4. Solid lines above each graph indicate results of Wilcoxon matched-pairs signed rank test. *P < .05, **P < .01, ***P < .001. ns, not statistically significant. PD, progression of disease.

Figure 5.

Molecular patterns of ibrutinib-resistant disease. A schematic representation of study findings. Sensitivity to ibrutinib is altered by histology and the presence of resistance-conferring mutations. Histologic transformation is an early event (<15 months in the study presented here), and can be de novo (clonally unrelated) or originate in the CLL clone (clonally related). Progressive CLL occurs later in the treatment course, and is frequently accompanied by BTK (red circles) and/or PLCG2 (blue circles) mutations. Multiple subclones can coexist, precede clinical progression by many months, and expand at different rates.