Comparison of Deep Whole Exome Versus Targeted Gene Sequencing for Assessment of Persistent Molecular Disease in Acute Myeloid Leukemia Samples
Authors: Slade Michael , O'Laughlin Michelle , Fulton Robert S , Duncavage Eric J. , Ley Timothy J , Jacoby Meagan A. , Spencer David H 
Published: February 2021

Background
There is growing evidence that genomics-based assessment of persistent molecular disease (PMD) may be a useful risk stratification tool for patients with acute myeloid leukemia (AML). However, there is no consensus about the optimal approach for detection and monitoring of leukemia-associated mutations (LAMs) for PMD testing. One approach is whole exome sequencing (WES), which provides a comprehensive assessment of the clonal architecture by identifying LAMs across the exome at diagnosis, which can then be measured in follow-up samples. Another approach is to use highly sensitive targeted gene sequencing (TGS) to detect persistent LAMs even at very low levels, although detection is limited to genes interrogated by the panel. Although both of approaches have been shown to predict outcome in retrospective studies, there are substantial differences in the number of LAMs identified and the limit of detection, and a head-to-head comparison has not yet been reported. Here, we use a defined cohort enrolled in a prospective clinical trial to compare PMD results from deep WES to error-corrected TGS.
Methods
Cohort: Patients were age 18-60 with de novo AML classified as intermediate risk by European Leukemia Net criteria, who achieved a morphologic remission after undergoing standard induction therapy. All patients were enrolled in a prospective clinical trial (NCT02756962).
Sequencing: Deep WES was performed using DNA from normal tissue (buccal swab or skin) and pre- and post-induction (~day 30) bone marrow (BM) samples to achieve an average coverage depth of ~600x. LAMs identified via paired tumor/normal analysis of the pre-induction sample were queried in the post-induction sample for assessment of PMD. PMD testing by TGS was performed using error-corrected sequencing of 40 genes recurrently mutated AML genes to an average error-corrected coverage depth of ~4500x.
Definitions: Based on previously published work, we used two separate variant allele frequency (VAF) cutoffs to define PMD. For WES, PMD+ was defined as ≥1 LAM with a VAF &gt;2.5% (Klco JAMA 2015). For TGS, PMD+ was defined as having ≥1 LAM with a VAF &gt;0.5% (Duncavage NEJM 2018). LAMs were sub-classified per Table 1. TGS mutations not identified as LAMs by WES at diagnosis were excluded from the primary analysis.
Results
31 patients were studied. LAMs are summarized in Table 1. 20 patients (65%) were PMD+ by WES after induction, and 22 patients (71%) were PMD+ by TGS. The concordance between WES and TGS was 81% (25/31) (Table 2). Two patients were PMD+ by WES only, due to the persistence of LAMs in the exome space, but not in the targeted panel. Four patients were PMD+ by TGS only, due to the presence of recurrent mutations at VAFs below the detection limit of WES (range: 0.59 - 1.90%). Two patients were PMD+ by both assays, but due to different mutations. All other patients who were PMD+ by both assays had at least one overlapping mutation.
Analysis of the mutations that persisted after therapy in both assays showed that 26% of patients (8/31) were PMD+ by TGS because of mutations in DNMT3A, TET2, or ASXL1 (i.e., "DTA" mutations). All of these patients were also PMD+ by WES, with 7 of the 8 patients having ≥1 additional non-DTA mutation (median: 3, range: 1 - 7). In an exploratory analysis, 22 additional mutations in 13 patients were identified by TGS that were not detected by WES on the diagnostic sample. This included two patients who were PMD-, but who had new mutations in TET2 and DNMT3A, respectively, likely representing selection for ancestral clones that were unrelated to the AML founding clone.
Conclusion
Concordance between WES and TGS-based PMD assessment was high. Discordant results were generally driven by non-recurrent mutations detected by WES, and low-level mutations detected by the high coverage, error-corrected TGS. Although isolated DTA mutations were common on TGS, WES analysis showed additional LAMs accompanied these variants in most cases, indicating the persistence of an ancestral leukemic clone that may provide useful prognostic information. We also observed new, low-level mutations that emerged after therapy in 42% of patients, some of which were not part of the leukemic clone identified at diagnosis. This indicates that use of highly sensitive PMD approaches may be challenging without pre-induction mutation testing, which is required to understand the relevance of markers of persistent molecular disease.

Disclosures
Jacoby: AbbVie: Research Funding; Takeda: Consultancy; Jazz Pharmaceuticals: Research Funding.