Real-Time Molecular Monitoring in Acute Myeloid Leukemia With Circulating Tumor DNA

Abstract

The clonal evolution of acute myeloid leukemia (AML), an oligoclonal hematological malignancy, is driven by a plethora of cytogenetic abnormalities, gene mutations, abnormal epigenetic patterns, and aberrant gene expressions. These alterations in the leukemic blasts promote clinically diverse manifestations with common characteristics of high relapse and drug resistance. Defining and real-time monitoring of a personalized panel of these predictive genetic biomarkers is rapidly being adapted in clinical setting for diagnostic, prognostic, and therapeutic decision-making in AML. A major challenge remains the frequency of invasive biopsy procedures that can be routinely performed for monitoring of AML disease progression. Moreover, a single-site biopsy is not representative of the tumor heterogeneity as it is spatially and temporally constrained and necessitates the understanding of longitudinal and spatial subclonal dynamics in AML. Hematopoietic cells are a major contributor to plasma cell-free DNA, which also contain leukemia-specific aberrations as the circulating tumor-derived DNA (ctDNA) fraction. Plasma cell-free DNA analysis holds immense potential as a minimally invasive tool for genomic profiling at diagnosis as well as clonal evolution during AML disease progression. With the technological advances and increasing sensitivity for detection of ctDNA, both genetic and epigenetic aberrations can be qualitatively and quantitatively evaluated. However, challenges remain in validating the utility of liquid biopsy tools in clinics, and universal recommendations are still awaited towards reliable diagnostics and prognostics. Here, we provide an overview on the scope of ctDNA analyses for prognosis, assessment of response to treatment and measurable residual disease, prediction of disease relapse, development of acquired resistance and beyond in AML.

Keywords: acute myeloid leukemia; cfDNA; circulating tumor DNA; ctDNA; genomic profiling; hematological malignancies; measurable residual disease; plasma cell free DNA.

FIGURE 1

Timeline of various milestones in the evolution of genomic classification of AML (Adapted from TCGA project Ley et al., 2013) and studies on circulating tumor DNA in AML. Initially, AML was classified by the French-American-British (FAB) Cooperative Group (1976) based on the cell lineage of leukemic cells and their differentiation status derived from the cell morphology as well as cytochemical staining of BM cells (Bennett et al., 1976). This approach had limitations in risk stratification in majority of AML cases. The discovery of recurrent cytogenetic abnormalities led to a novel classification system by the World Health Organization [WHO] (2001), which was revised in 2008 and later in the 2016 WHO Classification. Few research highlights of various studies on ctDNA analysis in AML disease monitoring are indicated in the timeline as a co-evolution with advances in AML genomics. ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ctDNA, circulating tumor-derived DNA; ed., edition; LOH, loss of helerozygosity; MRD, measurable residual disease; TS, targeted sequencing; WHO, World Health Organization.

FIGURE 2

The fractional abundance of KIT mutation in circulating tumor-derived DNA but not the levels of plasma cell free DNA represent residual AML better and may predict early relapse. Molecular monitoring of plasma cell-free DNA and bone marrow samples of AML patient was performed by droplet digital PCR at the indicated time points. Shown in the line graph are blast percentage (violet square), total plasma cfDNA concentration in nanograms per milliliter plasma (blue diamond), fractional abundance of KIT D816V mutation in cfDNA (red square), and bone marrow (green triangle) at baseline (BL = Day 0), MRD assessment (FU1 = Day 30), clinical remission (FU2 = 5 months), and after consolidation and relapse (FU3 = 6 months). BL, baseline; cDNA, complementary DNA; cfDNA, cell-free DNA; ctDNA, circulating tumor-derived DNA; FA, fractional abundance; FU, follow-up; MRD, measurable residual disease.

FIGURE 3

Comparison of technology platforms for analysis of cell-free DNA. The analytical workflow for cell-free DNA characterization and analysis are depicted showing key steps of isolation, quantitation, and various options of the technology platforms available for molecular analysis of circulating tumor-derived DNA depending on the target region and tumor burden. cfDNA, cell-free DNA; ctDNA, circulating tumor-derived DNA; CAPP-Seq, cancer personalized profiling by deep sequencing; CNVs, copy number variations; dsDNA (HS), double-stranded DNA (high sensitivity); LOD, limit of detection; PARE, parallel analyses of RNA ends; Safe-SeqS, safe-sequencing system; Tam-Seq, tagged amplicon deep sequencing.

FIGURE 4

Proposed integration of ctDNA profiling with AML diagnosis and disease monitoring. The current practice for AML diagnosis and disease monitoring is primarily dependent on the bone marrow sampling. A representation of the disease course is merged with the major time-points of investigation for evaluation of response to treatment (top panel). A critical time point post-chemotherapy needs to be defined when the disease is in occult stage and the MRD threshold is maintained (indicated by the gray downward arrow). Residual AML disease monitoring by serial sampling of circulating cfDNA analysis for patient-specific genetic alterations may prove informative when MRD threshold is breached (indicated in the middle panel). cfDNA profiling may provide several other applications in AML disease monitoring in real time as shown (bottom panel). BM, bone marrow; CBC, complete blood count; cfDNA, cell-free DNA; CR, clinical remission; MFC, multiparametric flow cytometry; MRD, measurable residual disease; PB, peripheral blood; SCT, stem cell transplantation; TMB, tumor mutation burden.