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Genetic and epigenetic intra-tumoral heterogeneity cooperate to shape the evolutionary course of cancer1. Chronic lymphocytic leukaemia (CLL) is a highly informative model for cancer evolution as it undergoes substantial genetic diversification and evolution after therapy2,3. The CLL epigenome is also an important disease-defining feature4,5, and growing populations of cells in CLL diversify by stochastic changes in DNA methylation known as epimutations6. However, previous studies using bulk sequencing methods to analyse the patterns of DNA methylation were unable to determine whether epimutations affect CLL populations homogeneously. Here, to measure the epimutation rate at single-cell resolution, we applied multiplexed single-cell reduced-representation bisulfite sequencing to B cells from healthy donors and patients with CLL. We observed that the common clonal origin of CLL results in a consistently increased epimutation rate, with low variability in the cell-to-cell epimutation rate. By contrast, variable epimutation rates across healthy B cells reflect diverse evolutionary ages across the trajectory of B cell differentiation, consistent with epimutations serving as a molecular clock. Heritable epimutation information allowed us to reconstruct lineages at high-resolution with single-cell data, and to apply this directly to patient samples. The CLL lineage tree shape revealed earlier branching and longer branch lengths than in normal B cells, reflecting rapid drift after the initial malignant transformation and a greater proliferative history. Integration of single-cell bisulfite sequencing analysis with single-cell transcriptomes and genotyping confirmed that genetic subclones mapped to distinct clades, as inferred solely on the basis of epimutation information. Finally, to examine potential lineage biases during therapy, we profiled serial samples during ibrutinib-associated lymphocytosis, and identified clades of cells that were preferentially expelled from the lymph node after treatment, marked by distinct transcriptional profiles. The single-cell integration of genetic, epigenetic and transcriptional information thus charts the lineage history of CLL and its evolution with therapy. A single-cell approach is used to follow the heritable stochastic changes to DNA methylation that occur in primary chronic lymphocytic leukaemia and healthy B cells, allowing the tracing of cell lineage histories and evolution during treatment with ibrutinib.

Introduction: Despite mapping of the mutational landscape in patients (pts) with chronic lymphocytic leukemia (CLL), there remains limited information regarding the functional or clinical impact of less common, recurrently mutated genes. Over the past several years, the influence of high-risk mutations has been reported. With the advent of readily available next generation sequencing (NGS), physicians receive a large amount of genomic data, many times without information regarding the clinical impact of less validated, mutations. Using results from a commercially available NGS platform, we identified a relatively high prevalence of mutations in the gene, FAT1, in treatment naïve (TN) CLL pts. FAT1 encodes a protein in the cadherin superfamily, important in regulating WNT signaling, with a role in tumor suppression. Aberrant mutated FAT1 expression has been associated with acute lymphoblastic leukemias with an intermediate clinical course. Loss of protein, through chromosomal deletions has been implicated in many solid tumors and associated with tumor progression. The majority of mutations arise in the cadherin repeats, previously shown to diminish antagonism of β-catenin, allowing its nuclear localization (Morris Nat Genetics 2013). In CLL, FAT1 mutations have been demonstrated to enrich in fludarabine refractory pts (10.3%) but with a relatively low prevalence in untreated pts (1.1%) (Messina, Blood 2014). Given a high prevalence of FAT1 mutations in our database and evidence suggesting mutated FAT1 contributes to tumor evolution, we investigated the clinical impact of clonal FAT1 mutations in pts with CLL. Methods: Patients were identified for inclusion if mutational analysis was performed prior to, or within 12 months of treatment start date. Only pts treated initially with a signal transduction inhibitor (STI) were included. Treated pts were either on protocol or received their STI as standard of care. All pts underwent whole exome profiling with a lymphoid specific NGS panel, including 75 genes (Genoptix Inc.). Analysis was performed on peripheral blood or bone marrow aspirates. The primary outcome was time to first treatment (TTFT). The log-rank test was used to compare Kaplan-Meir curves. Double-sided P values Results: In total we analyzed 172 pts, 118 (69%) remain on observation, 54 (31%), have been treated at data cutoff. Nineteen (11%) pts were found to be FAT1 mutated (mFAT1) and 153 (89%) were FAT1 wild type (wtFAT1). The median time from diagnosis to mutational profiling for the cohort was 35 months (range 0-301). We identified 21 total mutations, 17 unique (82% cadherin domain, 18% EGF like region). Recurrent mutations were identified, each occurring in 2 pts (p.A636T, p.P1614L, p.R1257Q, p.R2041H). One pt had an indel and 1 pt had an inframe deletion, the remaining were missense. We found no significant differences between groups in regards to age, IGVH mutation status or co-occurrence of high-risk mutations in NOTCH1, SF3B1, TP53 or ATM. Only 17p deletions occurred significantly more in mFAT1 pts (24%) vs. wtFAT1 (7%), p=0.04, other cytogenetic abnormalities were balanced. mFAT1 pts had significantly shorter TTFT, 50 versus 143 months respectively, p=0.02. We investigated if mFAT1 status could stratify IGVH mutated pts but found no differences in TTFT. Eleven mFAT1 pts have received treatment (82% ibrutinib, 18% acalabrutinib), 8 remain on observation. All mFAT1 pts have responded. Conclusions: Given the increasing use of rapidly available NGS testing, physicians receive genomic information with limited data regarding clinical impact. We identified a higher prevalence of FAT1 mutations in TN pts, than that previously reported. FAT1 mutations associated with deletion 17p but not other high-risk genetic mutations. Recurrent FAT1 mutations were identified and commonly found in the cadherin domain. mFAT1 pts have a significantly shorter TTFT than wtFAT1 pts. When treated with novel agents, there was no difference in response rates. These findings suggest FAT1 mutations in TN pts may be more common than previously reported and identify an intermediate risk for progression. Additional studies investigating the influence of recurrent FAT1 mutations and association with 17p deletions in TN pts are warranted. Disclosures Allan: Pharmacyclics: Speakers Bureau.