Mutational Analysis of Patients with Primary Resistance to Single-Agent Ibrutinib in Relapsed or Refractory Mantle Cell Lymphoma (MCL)

Introduction: Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton’s tyrosine kinase is highly effective in relapsed or refractory mantle cell lymphoma (MCL) patients with an ORR of 68% (Wang M, et al. N Engl J Med. 2013;369:507-516). Similar efficacy results were observed in a recent phase 2 study in MCL patients who progressed after rituximab containing chemotherapy and bortezomib therapy (SPARK study, NCT01599949). However, despite the very promising efficacy data, some patients with MCL do not respond to ibrutinib. Here, we report analyses of potential mechanisms associated with primary resistance to ibrutinib therapy in the SPARK study.

Methods: In this phase 2, international, multicenter, single-arm study, patients with MCL received 560 mg/day oral ibrutinib continuously until progressive disease (PD) or unacceptable toxicity. Patients who had PD at the first disease evaluation were considered to have primary resistant disease. DNA was extracted from baseline/pretreatment tumor samples (biopsy or CD19-enriched cells from PBMC) and enriched libraries were constructed with probesets specific for the coding region of 97 genes possibly involved in ibrutinib response and resistance, using the Ovation Target Enrichment system (NUGEN). Deep sequencing (150bp, single end reads) was performed on an Illumina HiSeq instrument . Sequences were aligned to the hg19 reference genome, variants were called using samtools and filters were applied to identify possible somatic mutations (minor allele frequency < 1% in dbSNP, > 5% and < 95% variant allele, > = 10 total reads).

Results: Twenty five (22.7%) of the 120 patients enrolled in this study had Independent Review Committee (IRC)-confirmed disease progression. In these primary treatment resistant patients, the median number of prior lines of systemic therapy was 3 (range 1-5 lines); 37% of patients had high risk MIPI score; 64% had bulky disease (longest diameter ≥ 5 cm), 52% had extranodal disease; 32% had bone marrow involvement and 20% had blastoid subtype. None of these baseline clinical parameters were found to be predictive for primary treatment resistance. The median duration of treatment was 1.54 months. Sequence data could be collected from 23 of the 25 patients, with an average of 9 million reads. After data filtering as described above, 27 genes were found with nonsynonymous variants in at least 2 or more patients. The majority of these variants were previously unreported in dbSNP or COSMIC databases. No mutations previously described in chronic lymphocytic leukemia (CLL) patients with acquired resistance to ibrutinib (BTK C481S, PLCg2 R665W) were seen in these patients, although one patient had a different mutation in PLCg2. Genes previously implicated in diffuse large B-cell lymphoma (DLBCL) pathogenesis (Pasqualucci L, et al. Nature Genetics. 2011;43:830-837) such as MLL2 and CREBBP were also found to be mutated in these patients. In addition, mutations in PIM1 and ERBB4 kinase genes were more frequent in patients with PD compared with those non-resistant to therapy. PIM1 kinase has been associated with increased proliferation, survival, and migration of tumor cells, and somatic mutations in this gene have been described in several cancers including hematologic malignancies. Interestingly, several of the mutations detected affect NF-kB signalling inhibition which is thought to contribute to ibrutinib’s mechanism of action. These results will be updated at the time of final presentation.

Conclusion: Sequence analyses were conducted on tumor DNA from the fraction of patients with primary resistance to ibrutinib treatment in this study. These studies have revealed a number of known and novel mutations, including genes involved in NF-kB signalling. Among others, the mutational status of PIM1 kinase and ERBB4 kinase genes may be of interest with respect to primary resistance to ibrutinib therapy in MCL.