Cell-free DNA for detection and monitoring of extramedullary AML relapse

Abstract

Isolated extramedullary manifestations (IEM) of acute myeloid leukemia (AML) are recurrent events, especially following allogeneic hematopoietic cell transplantation (alloHCT). To date, measurable residual disease (MRD) assessment for this difficult-to-treat patient cohort has not been established. In this study, we evaluated highly sensitive next-generation sequencing (NGS) of IEM-AML tumor and compared it with cell-free DNA (cfDNA) from plasma, as well as highly sensitive NGS analysis of bone marrow mononuclear cells (BMMC) and peripheral blood mononuclear cells (PBMC), in a cohort of 15 IEM-AML patients with 19 IEM-AML episodes. cfDNA demonstrated a superior representation of IEM-AML tumor mutations compared to BMMC or PBMC, with a median variant allele frequency (VAF) of 0.8% and a mutation detection rate of 62% (37 of 60 mutations), compared to a median VAF of 0.05% and detection rate of 27%, respectively (16 of 60 mutations, p < 0.01). Among 44 mutations identified in 14 IEM-AML relapse tumors, 30 mutations (68%) were known from initial diagnosis. Using diagnostic mutations from initial diagnosis for MRD analysis and detection of IEM-AML relapse, 16 of 17 IEM-AML relapse episodes were detected via cfDNA, whereas only 7 of 17 were identified using conventional analysis of BMMC or PBMC. Our findings demonstrate that cfDNA analysis from plasma effectively captures the molecular profile of IEM-AML. More than one-third of clinically relevant mutations were exclusively detected through cfDNA and were missed by conventional NGS-MRD of BMMC or PBMC. These results suggest that MRD monitoring using cfDNA offers a more comprehensive and sensitive approach to detecting IEM-AML relapse compared to standard methods.

Figure 1

IEM‐AML relapse mutations are represented in cfDNA. (A) Frequency of genes that were mutated in the IEM‐AML tumor tissue of 15 patients was assessed by highly sensitive NGS. (B) Ratio of IEM‐AML mutations and (C) IEM‐AML episodes that were detected by NGS analysis of cfDNA using the mutations from IEM‐AML tumor tissue. (D) Comparison of VAF (%) between IEM‐AML tumor and cfDNA. atyp, atypical; cfDNA, cell‐free DNA; eps., episodes; IEM, isolated extramedullary AML; inv, inversion; ITD, internal tandem duplication; TKD, tyrosine kinase domain; VAF, variant allele frequency.

Figure 2

Comparison of cfDNA versus MC‐derived DNA for detection of IEM‐AML. (A) Number of detected IEM‐AML mutations and (B) number of IEM‐AML episodes in the cfDNA and BM/PBMC compartments. (C) VAF comparison of matched mutations between cfDNA and BM/PBMC‐derived DNA for all IEM‐AML episodes. BM, bone marrow; cfDNA, cell‐free DNA; IEM, isolated extramedullary AML; eps, episodes; mut, mutations; MC, mononuclear cells; mut., mutated; PB, peripheral blood; VAF, variant allele frequency.

Figure 3

Clonal evolution of mutations from the initial diagnosis to IEM‐AML relapse. Number of mutations lost and gained at IEM‐AML relapse sorted by mutation classes, from 14 AML patients who developed an IEM‐AML relapse. Thrity of 44 (68%) mutations found in the IEM‐AML relapse tumor were known from the initial diagnosis. atyp, atypical; IEM, isolated extramedullary AML; Inv, inversion; ITD, internal tandem duplication; no., number; TFS, transcription factors; transd, transduction; TKD, tyrosine kinase domain.

Figure 4

Longitudinal NGS‐MRD analysis of mutations from IEM‐AML tumors using cfDNA and BM/PBMC. X‐axis: Time before (negative values) and after (positive values) the first IEM‐AML episode (First IEM‐AML = 0). Y‐axis: Number of patients included in our retrospective analysis. Green diamond and line: Both cfDNA and BM/PBMC‐derived DNA analysis positive. Blue diamond and line: Both cfDNA and BM/PBMC‐derived DNA analysis negative. Yellow diamond: cfDNA analysis positive, BM/PBMC‐derived DNA analysis negative. Yellow triangle: cfDNA analysis positive, BM/PBMC sample not available. Yellow line: cfDNA analysis positive. Red circle: Second IEM‐AML during the course of the disease. Black rectangle: Patient deceased. Triangle at the end: Patient still alive at last follow‐up. Gray line: No data available at that time point. Dashed lines: MRD‐status before first IEM‐AML. Solid lines: MRD‐status after 1st IEM‐AML. BM, bone marrow; cfDNA, cell‐free DNA; IEM, isolated extramedullary; MRD, measurable residual disease; N/A, not available; PB, peripheral blood.