Noninvasive Detection of Ibrutinib Resistance in Non-Hodgkin Lymphoma Using Cell-Free DNA
Authors: Scherer Florian , Kurtz David M. , Newman Aaron M , Craig Alexander , Stehr Henning , Zhou Li , Glover Cynthia , Kohrt Holbrook , Levy Ronald , Diehn Maximilian , Alizadeh Ash A. 
Published: October 2019

Abstract
Background: Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), plays an emerging role in targeted therapy of several lymphoid malignancies. Due to its activity in B-cell receptor (BCR) dependent human B-cell cancers, Ibrutinib is currently approved for the treatment of patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In addition, Ibrutinib responses can be seen in patients with follicular lymphoma (FL) and in a subset of patients with ABC- (activated B-cell-) like diffuse large B-cell lymphoma (DLBCL) (Wilson et al., Nat Med 2015; Fowler et al., ASH Annual Meeting, 2015). However, resistance mutations in BTK and other genes (e.g., PLCγ2)are a major cause of treatment failure and early identification of those aberrations remains challenging. Circulating tumor DNA (ctDNA) is an emerging biomarker with potential for disease monitoring as well as biopsy-free detection of evolving somatic aberrations. In this case study, we show that noninvasive genotyping of blood plasma allows early detection of resistance mutations duringIbrutinib therapy in a patient with relapsed/refractory follicular lymphoma associated with histological transformation.
Methods: We profiled two tumor lymph node (LN) biopsy specimens and 8 plasma samples before, during, and after Ibrutinib therapy of a 67-year-old man with relapsed/refractory FL with evidence of transformation. Preceding Ibrutinib, this patient received 6 lines of lymphoma treatment over a ~3 year disease course, including Rituximab monotherapy, Benadmustine/Rituximab, and R-CHOP. Despite achieving stable disease after 12 weeks, the patient experienced significant disease progression after additional 4 months on Ibrutinib (Figure 1). We profiled these 10 serial samples using CAPP-Seq, a capture-based targeted high-throughput sequencing (HTS) method (Newman et al., Nat Med, 2014), which allows the sensitive identification of emerging somatic aberrations in circulating tumor DNA.
Results: We detected the emergence of two independent BTK C481S resistance mutations in the blood plasma during Ibrutinib therapy, which were undetectable immediately before treatment. The major resistance clone harboring the BTK C481S A>T mutation (Figure 1, orange) was identified 2 months after the start of Ibrutinib, with allele frequencies as low as 0.03%. The second resistant subclone emerged 63 days later, carrying a BTK C481S somatic mutation with a substitution of C to G (Figure 1, green). Importantly, despite encoding an identical amino acid mutation, these two adjacent variants were never observed within the samectDNA molecule, demonstrating convergent evolution of independent resistantsubclones. We confirmed the existence of the two resistance mutations by tumor genotyping of a LN biopsy after clinical diagnosis of disease progression. Notably, while the minorsubclone completely disappeared under subsequentimmunochemotherapy with Rituximab/Bendamustine/Lenalidomide (RBL), the major resistance clone reappeared at the time of disease progression and the patient ultimately succumbed to his disease.
Conclusions: Resistance mutations in BTK C481S have been described in tumor cells of Ibrutinib-refractory patients with CLL and MCL, with in vitro evidence of responsiveness to BH3-mimetics and SYK inhibitors. This study, to our knowledge, is the first report of its noninvasive detection in the plasma of a patient with Non-Hodgkin lymphoma, with the subsequent partial eradication of a resistant subclone. Therefore, we envision that biopsy-free genotyping will allow early detection of lymphoma patients developing Ibrutinib resistance and will be incorporated into future clinical trials involving targeted therapies.
Figure 1 Figure 1.

Disclosures
Newman: Roche: Consultancy. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Diehn:Varian Medical Systems: Research Funding; Novartis: Consultancy; Roche: Consultancy; Quanticel Pharmaceuticals: Consultancy.