Abstract 3178: Deep sequencing of immunophenotypically distinct subsets in acute myeloid leukemia reveals reservoirs of genetically distinct subclones along a conserved differentiation trajectory.
Authors: Simonds Erin F. , Bendall Sean C. , Gedman Amanda L. , Levine Jacob H. , Davis Kara L. , Fienberg Harris G. , Jager Astraea , Amir El-ad D. , Radtke Ina , Fantl Wendy J. , Pe'er Dana , Downing James R. , Nolan Garry P. 
Published: August 2014

Acute myeloid leukemia (AML) can present with multiple concurrent subclones at diagnosis. Subclone-specific mutations may confer resistance to molecular-targeted drugs through loss of antigen expression or rewiring of intracellular signaling pathways, leading to relapse. Deep sequencing approaches improve the detection of rare subclones, but the ability to prospectively identify subclones (i.e. without relapse material) is limited, and the effects of subclone-specific mutations on phenotype are poorly understood. In the course of a broader study of oligoclonal pediatric AML patients, we focused investigation on a diagnosis bone marrow sample from a patient harboring 3 presumed subclones (two with distinct NRAS-G12D, NRAS-G13D mutations) as determined by whole-genome sequencing (WGS). We employed a combination of 31-parameter mass cytometry, deep sequencing, FACS sorting, and computational modeling to produce a detailed profile of the subclonal genotypes and phenotypes in this patient.

The 3 anticipated subclones did not correlate with a clear subset of surface markers in the mass cytometry analysis. Instead, we observed a single continuous ‘differentiation trajectory’ from progenitor-like to monocyte-like blasts. To dissect this trajectory, 7 distinct myeloid/progenitor subsets were FACS-sorted, plus T/B cells as a non-leukemic control. For each subset we performed capture-based deep sequencing of 307 tumor-specific variants (Tier 1: 13; Tier 2: 33; Tier 3: 261; median depth: 1420x). Shifts in allele frequencies among the FACS-sorted subsets provided critical information to a Bayesian mixture modeling algorithm, allowing identification of 5 subclones, as opposed to the 3 subclones anticipated by WGS. The inferred subclonal genotypes were validated and further refined by targeted single-cell Sanger sequencing of multiple Tier 3 loci.

Although all 5 subclones were present throughout the differentiation trajectory, some were enriched in certain phenotypic states or ‘reservoirs’. For example, the NRAS-G12D subclone was enriched in a progenitor-like subset, but its daughter subclone, which harbored an additional mutation in RAC2 (implicated in HSC engraftment), was enriched in the more differentiated subsets, suggesting an opposing effect. Notably, the T/B cell population harbored 2 tumor-specific Tier 1 mutations at &gt;15% allele frequency, suggesting its presence in a preleukemic, multilineage-competent HSC.

Taken together, subclone-specific mutations appear to skew cells toward either progenitor or mature phenotypes, but the developmental trajectory enforced by parental mutations is resistant to change. Furthermore, characterization of subclones is possible at diagnosis, and may improve the selection of targeted therapies.

*Contributed equally: EFS, SCB & ALG

Citation Format: Erin F. Simonds, Sean C. Bendall, Amanda L. Gedman, Jacob H. Levine, Kara L. Davis, Harris G. Fienberg, Astraea Jager, El-ad D. Amir, Ina Radtke, Wendy J. Fantl, Dana Pe'er, James R. Downing, Garry P. Nolan. Deep sequencing of immunophenotypically distinct subsets in acute myeloid leukemia reveals reservoirs of genetically distinct subclones along a conserved differentiation trajectory. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3178. doi:10.1158/1538-7445.AM2013-3178