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Whole Exome Sequencing of Chronic Lymphocytic Leukemia

The somatic genetic basis of chronic lymphocytic leukemia (CLL), a common and clinically heterogenous adult leukemia, remains poorly understood. Massively parallel sequencing technology now provides a method for systematic discovery of genetic alterations that underlie disease, and for uncovering new therapeutic targets and biomarkers. In study version 2, we presented a dataset consisting of DNA sequencing from 169 CLL samples (with matched germline controls). Samples were collected from patients displaying a wide range of characteristics representing the broad clinical spectrum of CLL.

Understanding the mutational landscape of CLL provides a starting point for systematic analyses to address fundamental questions in CLL, including how mutated genes alter cellular networks and phenotypes, and thereby contribute to disease heterogeneity.

Intratumoral heterogeneity plays a critical role in tumor evolution. To define the contribution of DNA methylation to heterogeneity within tumors, we performed genome-scale bisulfite sequencing of >100 primary chronic lymphocytic leukemias (CLLs; data presented in study version 3). Compared with 26 normal B cell samples, CLLs consistently displayed higher intrasample variability of DNA methylation patterns across the genome, which appears to arise from stochastically disordered methylation in malignant cells. Transcriptome analysis of bulk and single CLL cells revealed that methylation disorder was linked to low-level expression. Disordered methylation was further associated with adverse clinical outcome. We therefore propose that disordered methylation plays a similar role to that of genetic instability, enhancing the ability of cancer cells to search for superior evolutionary trajectories.

Samples from this study were included in the CLL-map project, which integrated genomic, transcriptomic and epigenomic data from 1,148 patients to portray the landscape of genetic mutations and molecular phenotypes in CLL, and assess their association with patient outcome.

We conducted high-throughput dynamic BH3 profiling, a functional assay that measures the initiation of apoptotic signaling after ex vivo exposure to drugs, for interrogation of >60 CLL samples with exome, transcriptome and methylome sequencing. FDA-approved 42 drugs were used in this screen, which revealed associations between ex vivo drug response with molecular features. After augmenting study version 4 with additional RNA and methylome data, the entire dataset now includes RNA sequencing of primary CLLs from 82 subjects and of B-cells from 6 healthy donors, and methylome sequencing of primary CLLs from 121 subjects and of samples from 11 healthy donors.