Prevalence of BTK and PLCG2 mutations in a CLL cohort still on ibrutinib after three years

Ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, has shown efficacy in treating chronic lymphocytic leukemia (CLL) in both first-line and the relapsed/refractory setting. Studies performing mutational analyses after-acquired ibrutinib resistance have observed an association between mutations in the BTK and/or phospholipase Cγ2 (PLCG2) genes and CLL progression.^1,2 Despite this suggestion, information surrounding mutations for patients with CLL, who are still on ibrutinib after three years is limited,² and so there is still doubt as to whether these mutations directly cause ibrutinib resistance.³

As ibrutinib became available in France in 2014 under an early access program (EAP), a study conducted by the French Innovative Leukemia Organization (FILO) allows for the evaluation of prolonged use of ibrutinib in a real-life cohort. Anne Quinquenel, from the Hôpital Robert Debré, Université Reims Champagne-Ardenne, Reims, FR, and colleagues, conducted a study to investigate patients from the FILO study, who were still on ibrutinib after three years.

Study design

Fresh blood samples were taken from patients who began ibrutinib via the French EAP between February 2014 and April 2015 and were still on ibrutinib after three years. Flow cytometry analyses on the samples determined the presence of minimal residual disease (MRD) at a level of < 1 CLL cell per 10,000 lymphocytes (< 10^-4). Samples that displayed a CLL clone of > 0.5 x 10⁹/L, were profiled by next-generation sequencing (NGS).

NGS was performed for the following genes:

• BTK
• PLCG2
• TP53
• NOTCH1
• SF3B1
• XPO1
• RPS15
• ATM
• POT1
• BIRC3
• FBXW7

Samples showing BTK C481S with variant allele frequency (VAF) < 10% through NGS, were confirmed by droplet digital polymerase chain reaction (ddPCR).

Patients were then followed for CLL progression (assessed by the local FILO investigator) and treatment outcomes, including event-free survival (EFS), defined as a new CLL treatment or death, and overall survival (OS).

Key findings

Of the initial cohort of 204 patients who received ibrutinib via the EAP at 29 FILO centers in France, 31% (63) were still receiving ibrutinib after at least three years. Out of the 63 patients still on ibrutinib, 57 patients provided fresh blood samples, with the median time between the initiation of ibrutinib and sample collection being 3.5 years (range, 2.8–4.2).

Most of the 57 patients who provided samples had adverse prognostic factors. , Out of these 57 patients, 54 were previously treated (median of 2 lines), typically with alemtuzumab or immunochemotherapy. All 57 patients had detectable MRD, with 13% (7/55 with response data) with a partial response with lymphocytosis.

Mutational profiling

Of the samples tested, 30 exhibited a CLL clone > 0.5 x 10⁹/L and were profiled using NGS:

• Half (15/30) of the samples had a TP53 mutation
• At least one BTK mutation was observed in 17/30 (57%) patients and the VAF range was wide (0.2−73%).
  • Of the 17 patients with BTK mutations, most (14/17) had a single mutation, with no statistically significant associations between the presence of a BTK mutation and the number of previous treatment lines (0 or 1 vs >2), need for dose interruptions/reductions, or presence of other mutations
• At least one PLCG2 mutation occurred in 13% (4/30)
  • 3/4 patients had two PLCG2 mutations
  • All 4 patients also had a BTK mutation and a TP53 mutation

Follow-up for CLL progression

At a median follow-up of 8.5 months from sample collection, 38 patients (19% of initially treated patients) continued treatment with ibrutinib. The researchers highlighted the drop from 28% patients at sample collection to 19% and suggested that this could be due to patients experiencing CLL progression between the third and fourth year on treatment.

• Of the 27 patients with CLL clones <0.5 x 10⁹/L, four subsequently progressed
  • Two patients were mutationally profiled at relapse
    • One patient with BTK mutation only
    • One patient with both BTK and PLCG2 mutations and the
  • Of the 30 patients with a CLL clone ≥0.5 x 10⁹/L, results showed that the presence of a BTK mutation was significantly associated with CLL progression (p= 0.0005)
  • Of the 17 patients with a BTK mutation:
    • 14 patients progressed:
      • Three patients remained on ibrutinib
      • Nine patients received venetoclax (5/7 evaluable had an objective response)
      • Two patients died without further treatment
    • Of the 13 patients without a BTK mutation:
      • Two patients progressed:
        • One patient received venetoclax
        • One patient died without further treatment
      • EFS was significantly shorter in patients with a BTK mutation (p= 0.0380), however, no difference was observed in OS.

Conclusion

This real-life mutational profile study of patients with CLL, still using ibrutinib after three years, showed that over half of patients with a CLL clone ≥0.5 x 10⁹/L had a BTK mutation, that this mutation conferred a greater likelihood of subsequent CLL progression.

The findings of the study support that mutational analysis should be considered in patients receiving ibrutinib with residual clonal lymphocytes. The researchers also put forward the case for further clinical trials to evaluate the use of other treatments for patients with BTK mutations.