Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition

ARTICLE Received 16 Sep 2015 | Accepted 12 Apr 2016 | Published 20 May 2016 DOI: 10.1038/ncomms11589 OPEN Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition Jan A. Burger1,*, Dan A. Landau2,3,4,5,*, Amaro Taylor-Weiner2,*, Ivana Bozic6,7,*, Huidan Zhang8,9,10,*, Kristopher Sarosiek11, Lili Wang11, Chip Stewart2, Jean Fan12, Julia Hoellenriegel1, Mariela Sivina1, Adrian M. Dubuc13, Cameron Fraser11, Yulong Han14, Shuqiang Li15, Kenneth J. Livak15, Lihua Zou2, Youzhong Wan11, Sergej Konoplev16, Carrie Sougnez2, Jennifer R. Brown11, Lynne V. Abruzzo16, Scott L. Carter2, Michael J. Keating1, Matthew S. Davids11, William G. Wierda1, Kristian Cibulskis2, Thorsten Zenz17, Lillian Werner18, Paola Dal Cin13, Peter Kharchencko12, Donna Neuberg18, Hagop Kantarjian1, Eric Lander2, Stacey Gabriel2, Susan O’Brien1, Anthony Letai11, David A. Weitz8, Martin A. Nowak6,7, Gad Getz2 & Catherine J. Wu2,11,19 Resistance to the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing transdifferentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance. 1 Department of Leukemia, MD Anderson Cancer Center, Houston, Texas 77030, USA. 2 Broad Institute, Cambridge, Massachusetts 02142, USA. 3 Department of Medicine, Weill Cornell Medicine, New York, New York 10065, USA. 4 Department of Physiology and Biophysics, Weill Cornell Medicine, New York, New York 10065, USA. 5 New York Genome Center, New York, New York 10013, USA. 6 Department of Mathematics, Program for Evolutionary Dynamics, Harvard University, Cambridge, Massachusetts 02138, USA. 7 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138, USA. 8 Department of Physics, School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, USA. 9 Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, China Medical University, Shenyang 110001, China. 10 Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110001, China. 11 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA. 12 Center for Biomedical Informatics, Harvard Medical School, Boston Massachusetts 02115, USA. 13 Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA. 14 Bioinspired Engineering and Biomechanics Center, Xi’an Jiaotong University, Xi’an 710049, China. 15 Fluidigm Corporation, South San Francisco, California 94080, USA. 16 Department of Hematopathology, MD Anderson Cancer Center, Houston, Texas 77030, USA. 17 National Center for Tumors and German Cancer Research Center (DKFZ), 69121 Heidelberg, Germany. 18 Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 19 Department of Medicine, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.A.B. (email: ) or to C.J.W. (email: ). NATURE COMMUNICATIONS | 7:11589 | DOI: 10.1038/ncomms11589 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11589 B -cell receptor (BCR) signalling is a critical growth and survival pathway in several B-cell malignancies, including chronic lymphocytic leukaemia (CLL)1. BCR signalling can be abrogated by novel kinase inhibitors that target the BCR-associated kinases SYK2, Bruton’s tyrosine kinase (BTK)3 and PI3Kd (ref. 4). The BTK inhibitor ibrutinib is a small molecule that inactivates BTK through irreversible covalent binding to Cys-481 within the ATP-binding domain of BTK5. In a recent trial in patients with relapsed/refractory CLL, ibrutinib induced an overall response rate of 71% and an estimated progression-free survival rate of 75% after 26 months of therapy3. However, a small fraction of patients develop progressive CLL after initially responding to ibrutinib3. Among these, patients carrying BTK mutations at the ibrutinib-binding site (C481S) or affecting the BCR-signalling-related molecule PLCg2 (R665W, L845F and S707Y) were recently highlighted6,7. The ability of cancer cells to evolve and adapt to targeted therapies is a challenge that limits treatment success and durability of responses. Whole-exome sequencing (WES), along with analyses of clonal heterogeneity and clonal evolution in CLL, can provide insight into emergence and expansion of subclones that carry driver mutations (for example, SF3B1 and TP53) under therapeutic pressure8. To determine the pattern of clonal evolution in ibrutinib-resistant CLL patients, we performed a longitudinal genomic investigation of five CLL patients who achieved partial remissions and later experienced disease progression. Here we demonstrate that ibrutinib therapy favours the selection and expansion of CLL subclones, carrying del(8p) with additional driver mutations in three different patients. Our studies provide insight into dynamic changes in clonal architecture during ibrutinib therapy, as well as potential alternative mechanisms of resistance. These findings are of particular interest, given the increasing clinical use of this BTK inhibitor. Results CLL relapse following ibrutinib therapy in Patients 1–4. Ibrutinib therapy generally is highly effective, and responses are durable in the vast majority of patients. However, some patients who initially respond the CLL eventually relapse because of the development of resistance, and presence of del(17p), especially in combination with other complex cytogenetic alterations, has been reported to predispose CLL patients for development of resistance9. In patients with relapsed/refractory CLL, the reported rate of ibrutinib resistance at 26 months was 13% (ref. 3) or 7.5% at a median follow-up of 16.8 months (ref. 10). Five patients from these early clinical (...truncated)