Abstract PR04: Clonal landscapes of hematologic malignancies redefined by ultrasensitive duplex sequencing
Authors: Jake Higgins, Lindsey N. Williams, Charles C. Valentine, Gabriel Pratt, Rashmi Kanagal-Shamanna, Nicholas J. Short, Masumi Ueda, Rainer Storb, Jerald P. Radich, Jesse J. Salk
Journal: Clinical Cancer Research (2020)

Abstract Hematologic malignancies (HM) are driven by clonal evolution—from preneoplastic clones through acquired drug resistance. Clonal hematopoiesis of indeterminate potential (CHIP) is a process whereby healthy individuals accumulate low-frequency clones with driver gene mutations. CHIP is associated with increased risk of HM but is challenging to detect. Measurable residual disease (MRD) after therapy in acute myeloid leukemia (AML) and other HM is a strong predictor of relapse; however, current MRD assays either have limited sensitivity or are narrowly applicable. Tyrosine kinase inhibitors (TKI) target BCR-ABL1 in chronic myeloid leukemia (CML) and Ph+ acute lymphocytic leukemia (ALL), yet TKI-resistance (TKI-R) mutations can drive relapse. Early detection of MRD and TKI-R mutations can inform important clinical interventions. Next-generation sequencing (NGS) can detect HM subclones, but not below a frequency of approximately 1%. Common error correction methods have difficulty detecting mutations below 0.1%. Duplex sequencing (DS) is a sophisticated NGS error correction assay capable of detecting individual mutations down to ultralow frequencies of at least 0.001% with a background error frequency below 1 in 10 million bases. We used DS for three clinically relevant HM applications: (1) CHIP: We performed DS on DNA from 10 hematopoietic cell transplant (HCT) donor/recipient pairs 7-46 years post-HCT. We targeted 29 genes recurrently mutated in AML, many of which have been described in CHIP. DS error-corrected depth up to 45,402x was generated. We identified dozens of clonal exonic variants (CEV) in every donor and recipient, most nonsynonymous, down to variant allele frequency (VAF) of 5.7E-5. The majority of the 154 shared donor/recipient CEVs were present at VAF &amp;lt;0.1%, with many increasing as well as decreasing in the recipient. (2) AML MRD: Dozens of genes are recurrently mutated in AML, with multiple mutations per patient. To model MRD, we mixed mutant cell line DNA into normal DNA from a healthy young control. 9 unique mutations were titrated to predicted VAF of 1%-0.001%. Replicate DS libraries generated over 1,000,000x total molecular depth with a targeted capture panel. All mutations were identified close to expected values with r2= 0.97. (3) HM drug resistance: We performed DS on Ph+ ALL samples in TKI-induced remission. The relapse TKI-R mutation was detected in samples banked 1-5 months prior to relapse at VAFs from 0.1%-1%. DS analysis of genomic DNA prior to TKI therapy demonstrated extremely low prevalence of resistance mutations in TKI-naïve patients, and cDNA analysis revealed extensive false positives. DS is a flexible, ultrasensitive technology for detection of low-frequency mutations in HM. DS redefines the landscape and prevalence of CHIP and reveals complex HCT donor/recipient dynamics. DS can detect MRD mutations at levels below 1/100,000 and identify TKI-R mutations months prior to relapse, which will better predict relapse and inform personalized therapy options. This abstract is also being presented as Poster A48. Citation Format: Jake Higgins, Lindsey N. Williams, Charles C. Valentine, Gabriel Pratt, Rashmi Kanagal-Shamanna, Nicholas J. Short, Masumi Ueda, Rainer Storb, Jerald P. Radich, Jesse J. Salk. Clonal landscapes of hematologic malignancies redefined by ultrasensitive duplex sequencing [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR04.