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Clinical Trial
. 2017 Sep 1;127(9):3484-3495.
doi: 10.1172/JCI91964. Epub 2017 Aug 21.

Ultrasensitive mutation detection identifies rare residual cells causing acute myelogenous leukemia relapse

Brian Parkin  1 , Angelina Londoño-Joshi  2 , Qing Kang  1 , Muneesh Tewari  1   3   4   5 , Andrew D Rhim  2 , Sami N Malek  1
Affiliations
Clinical Trial

Ultrasensitive mutation detection identifies rare residual cells causing acute myelogenous leukemia relapse

Brian Parkin et al. J Clin Invest. .

Abstract

Acute myelogenous leukemia (AML) frequently relapses after complete remission (CR), necessitating improved detection and phenotypic characterization of treatment-resistant residual disease. In this work, we have optimized droplet digital PCR to broadly measure mutated alleles of recurrently mutated genes in CR marrows of AML patients at levels as low as 0.002% variant allele frequency. Most gene mutations persisted in CR, albeit at highly variable and gene-dependent levels. The majority of AML cases demonstrated residual aberrant oligoclonal hematopoiesis. Importantly, we detected very rare cells (as few as 1 in 15,000) that were genomically similar to the dominant blast populations at diagnosis and were fully clonally represented at relapse, identifying these rare cells as one common source of AML relapse. Clinically, the mutant allele burden was associated with overall survival in AML, and our findings narrow the repertoire of gene mutations useful in minimal residual disease-based prognostication in AML. Overall, this work delineates rare cell populations that cause AML relapse, with direct implications for AML research directions and strategies to improve AML therapies and outcome.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. VAF detected in CR bone marrow samples categorized per gene.
The mean of duplicate measurements is depicted. The VAF percentage is on a log10 scale. VAFs were not corrected for genomic copy number aberrations (aCNAs), as these are rare at sites of mutated genes in AML (39).
Figure 2
Figure 2. Concordant mutant gene VAFs in CR bone marrow from 30 AML patients with 2 or more measured genes.
The mean of duplicate measurements is depicted. The VAF percentage is on a log10 scale. Individual patients study IDs are displayed on the x axis.
Figure 3
Figure 3. Discordant mutant gene VAFs in CR bone marrow samples from 38 AML patients with 2 or more measured genes.
Patients with discordant mutant gene VAFs demonstrating the common presence of oligoclonal aberrant hematopoiesis. DNMT3A, ASXL1, TET2, and IDH1 frequently persisted at greater than 1% VAF in CR. The mean of duplicate measurements is depicted. The VAF percentage is on a log10 scale.
Figure 4
Figure 4. Persistent rare cells that are genomically similar to the presentation disease blasts constitute a common source of relapsed AML.
Pre-therapy and relapse VAFs were measured with deep-coverage NGS (or ddPCR for FLT3-ITD), and CR VAFs were measured with ddPCR. In each panel, the graphs display VAFs of longitudinal measurements of mutationsF. (A–N) Examples of patients with discordant mutations demonstrating fully clonal representation at relapse of cell populations containing mutations with the lowest detected VAF in CR. (N and O) Two patients had mutations not detected in CR that were also not detected at relapse. The mean of duplicate measurements is depicted. The VAF percentage is on a log10scale. The VAFs of genes present on the X chromosome (specifically BCOR and STAG2) were corrected for male patients by a factor of 2 to accurately depict clonal composition of pre-therapy and relapse specimens.
Figure 5
Figure 5. Association of mutant gene VAF with remission duration.
(A) AML patients were grouped by remission duration for those who relapsed within less than 1 year from CR; relapsed after 1 year from CR; or had a durable remission for more than 1,000 days from CR. The VAF of mutated genes measured per patient is arranged by remission duration (left, shortest; right, longest). DNMT3A, ASXL1, TET2, and STAG2 mutations persisted at high VAF in patients who nevertheless experienced a durable remission. The mean of duplicate measurements is depicted. Patients who received alloSCT during the first CR are denoted with an asterisk. (B and C) Comparison of the median VAF for the lowest measured VAF (B) and the highest measured VAF (C) (excluding DNMT3A) in each patient, respectively, for various remission duration groups. The mean of duplicate measurements is depicted. Horizontal lines represent the geometric mean for each group. #P < 0.01; pair-wise comparisons were made using a non-Gaussian, unpaired, 2-tailed Mann-Whitney U test, corrected for multiple hypotheses testing using Bonferroni’s method. The VAF percentage is on a log10 scale.
Figure 6
Figure 6. Kaplan-Meier analyses of remission duration and survival based on mutant gene VAF in CR detected with ddPCR.
(A and B) RFS using the lowest measured VAF per patient categorized by 10-fold decreases in VAF or by dichotomous cutoff, respectively. (C and D) OS using the lowest measured VAF per patient categorized by 10-fold decreases in VAF or by dichotomous cutoff, respectively. (E and F) RFS using the highest measured VAF (excluding DNMT3A) per patient categorized by 10-fold decreases in VAF or by dichotomous cutoff, respectively. (G and H) OS using the highest measured VAF (excluding DNMT3A) per patient categorized by 10-fold decreases in VAF or by dichotomous cutoff, respectively. P < 0.01, by log-rank test for trend (A, C, E, and G) and by log-rank Mantel-Cox test (B, D, F, and H).
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