Developmental subtypes assessed by DNA methylation-iPLEX forecast the natural history of chronic lymphocytic leukemia

Brian Giacopelli, Qiuhong Zhao, Amy S. Ruppert, Akwasi Agyeman, Christoph Weigel, Yue-Zhong Wu, Madelyn M. Gerber, Kari G. Rabe, Melissa C. Larson, Junyan Lu, James S. Blachly, Kerry A. Rogers, William G. Wierda, Jennifer R. Brown, Kanti R. Rai, Michael Keating, Laura Z. Rassenti, Thomas J. Kipps, Thorsten Zenz, Tait D. Shanafelt, Neil E. Kay, Lynne V. Abruzzo, Kevin R. Coombes, Jennifer A. Woyach, John C. Byrd and Christopher C. Oakes

Key Points

  • The Me-iPLEX assay is an accurate and robust method for efficiently measuring multiplexed panels of DNA methylation sites.

  • Me-iPLEX-determined epitypes stratifies risk regardless of CLL disease progression or treatment with either chemoimmunotherapy or ibrutinib.


Alterations in global DNA methylation patterns are a major hallmark of cancer and represent attractive biomarkers for personalized risk stratification. Chronic lymphocytic leukemia (CLL) risk stratification studies typically focus on time to first treatment (TTFT), time to progression (TTP) after treatment, and overall survival (OS). Whereas TTFT risk stratification remains similar over time, TTP and OS have changed dramatically with the introduction of targeted therapies such as the Bruton's tyrosine kinase inhibitor, ibrutinib. We have shown genome-wide DNA methylation patterns in CLL are highly associated with phenotypic differentiation and patient outcomes. Here we developed a novel assay, termed methylation-iPLEX (Me-iPLEX), for high-throughput quantification of targeted panels of single CpGs from multiple independent loci. Me-iPLEX was used to classify CLL samples into one of three known epigenetic subtypes (epitypes). We examined the impact of epitype in 1,286 CLL patients from four independent cohorts representing a comprehensive view of CLL disease course and therapies. We found epitype significantly predicted TTFT and OS among newly diagnosed CLL patients. Additionally, epitype predicted TTP and OS with two common CLL therapies, chemoimmunotherapy (CIT) and Ibrutinib. Epitype upheld significance after stratifying by biologically-related biomarkers, IGHV-mutational status and ZAP70 expression, as well as other common prognostic markers. Furthermore, among several biological traits enriched between epitypes, we found highly-biased immunogenetic features, including IGLV3-21 usage in the poorly-characterized intermediate-programmed (IP)-CLL epitype. In summary, Me-iPLEX is an elegant method to assess epigenetic signatures, including robust classification of CLL epitypes that independently stratify patient risk at diagnosis and time of treatment.

  • Submitted March 7, 2019.
  • Revision received July 10, 2019.
  • Accepted July 3, 2019.