Abstract Introduction: The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is the first approved therapy for Waldenström's macroglobulinemia (WM), and is highly active in both treatment-naive and relapsing or refractory patients. Although ibrutinib is highly active in WM patients, disease progression can occur. Acquired mutations in BTK at the binding site of ibrutinib (Cys481), or in the protein immediately downstream of BTK, the phospholipase PLCG2, have been identified in half of progressing WM patients on ibrutinib (Xu et al, Blood 2017). However, not all ibrutinib resistant patients harbor these alterations, suggesting that there are other causes of disease progression on ibrutinib. The aim of this study was to identify alternative molecular mechanisms that can drive ibrutinib resistance. Patients and Methods: Five WM patients who progressed while on ibrutinib were studied. Tumor DNA samples at diagnosis, relapse, and germline DNA were available in three patients. For the remaining two, relapse and germline samples were sequenced. DNA was extracted from CD19-selected bone marrow mononuclear cells from patients. Non-CD19 cells from peripheral blood were used as germline controls. Samples were sequenced using whole exome sequencing. Data were analyzed following the Broad Institute's GATK Best Practice Guidelines. Small variants were analyzed using Strelka and MuTect2. Somatic structural variants were detected using Manta, and copy number alterations were called using Control-FREEC. Results: Copy number analysis identified deletions in chromosome 6q in all patients, becoming homozygous in two of them at relapse. In another patient, the homozygous deletion was already present at baseline in a third of the tumor population, and increased at relapse. No other recurrent copy number alterations were detected, though in two patients multiple deletions or gains involving large chromosomal regions were observed. Regarding small variants, relapse samples showed a high proportion of acquired mutations detected at relapse only (median 85%, range 79%-88%) compared to persistent mutations detected at both baseline and relapse (median 15%, range 12%-21%). Three out of the five patients harbored mutations in BTK (two with p.Cys481Ser in the kinase domain and the other a p.Thr62Asn in the PH domain). In patients with a BTK mutation, other alterations were observed in genes related to the B-cell receptor pathway including PLCG2 p.Y495H; CD79B p.D33Y; LYN p.A2Stop and LYN p.A139T. In patients without BTK mutations, we identified several mutations with a putative role in ibrutinib resistance that emerged at relapse including AIP4, an E3 protein-ubiquitin ligase whose substrates are CXCR4, LYN or SYK, in which a recurring p.A646S mutation was observed in two patients; RNF19B, an E3 protein-ubiquitin ligase involved in the cytolytic activity of natural killer cells and cytotoxic T-cells, in which a p.R30G mutation was observed in two patients; FCRL3, an Fc receptor-like 3 that differentially modulates innate immune signaling in B cells, in which a p.E694Q was observed in one patient; Mutations in the negative regulators of Toll-like receptors signaling were observed that included DOK2 p.Y345Stop, and TOLLIP p.M242R and p.R228H which were each observed once in separate patients. Mutant allele frequency clustering analysis by k-means reflected a linear pattern of evolution from baseline to relapse, in which most persistent alterations maintained the same allele frequency (median 18%), and acquired mutations were present in a small proportion of tumor cells (median 9.3%, p<0.01). Conclusions: Our findings depict uniform deletion of 6q, including homozygous loss of 6q, which encompass key regulators of BTK, BCL2, and NFKB; as well as emergence of novel gene mutations, including recurring mutations in E3 ubiquitin ligases, innate immune signaling, and Toll receptor/MYD88 pathway regulators as significant genomic alterations that accompany disease progression on ibrutinib in WM patients. Disclosures Castillo: Genentech: Consultancy; Beigene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Consultancy, Research Funding. Treon:Johnson & Johnson: Consultancy; BMS: Research Funding; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding. Hunter:Pharmacyclics: Consultancy.