Clonal Evolution and Changes in Two AML Patients Detected with A Novel Single-Cell DNA Sequencing Platform

Liwen Xu,Maurizio Pellegrino,Everett Meyer,Robert Durruthy-Durruthy,Dennis J. Eastburn,Omid Shah,James Zehnder

doi:10.1038/s41598-019-47297-z

Liwen Xu, Maurizio Pellegrino + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-47297-z

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Abstract

Next-generation sequencing (NGS) is used to detect gene variants in genetically complex cell populations of cancer patient samples. Traditional bulk analysis can only provide average variant allele frequencies of the targeted genes across all sampled cells. It fails to resolve mutational co-occurrences and may miss rare cancer cells. Genome analysis at the single cell level offers the opportunity to more fully resolve clonal architecture. Peripheral blood mononuclear cells were sampled from acute myeloid leukemia patients longitudinally and single-cell DNA sequencing libraries were generated with a novel droplet-based microfluidics approach. Molecular profiling of single nucleotide variants across thousands of cells revealed genetic chimerism in patients after bone marrow transplantation (BMT). Importantly, hierarchical clustering analysis of single nucleotide variants (SNVs) uncovered a distinct oncogenic clone of cells carrying mutated tumor-suppressor and/or oncogene(s). This novel single-cell DNA sequencing approach enabled precise monitoring of engraftment and revealed clonal evolution of oncogenic cells during the progression and treatment of the disease.

Highlights

In this study, we used a novel two-step droplet microfluidics approach that enables to profile genomic alterations across thousands of cells in targeted and automated fashion ref.[15]
The single-cell platform we used in this study facilitates a novel two-step droplet microfluidics approach to detect genomic DNA alterations (single nucleotide variants (SNVs) and short indels) across thousands of cells at single cell level in targeted, scalable and automated fashion
Amplified products were prepared with conventional sequencing library chemistry, single-cell sequencing libraries were sequenced on a MiSeq instrument and the data was processed and analyzed with Mission Bio’s cloud-based analysis software platforms (Supplementary Figure 1)

Summary

Introduction

We used a novel two-step droplet microfluidics approach that enables to profile genomic alterations across thousands of cells in targeted and automated fashion ref.[15]. Using the Tapestri Platform we analyzed peripheral blood mononuclear cells (PBMCs) from two AML patients longitudinally at three distinct time-points: before bone marrow transplant (pre-BMT), after bone marrow transplant (post-BMT) and at AML relapse www.nature.com/scientificreports/. The single-cell DNA-sequencing (DNA-seq) data allowed us to directly assess donor/host chimerism using the individuals’ unique genotype signatures as genetic proxies. We successfully identified all bulk DNA-seq verified mutations in the single-cell DNA-seq data and showed that number and frequency of variants corroborated bulk NGS data. We identified a unique clone of oncogenic cells that can’t be detected with conventional bulk sequencing. Comparison of clone number and size across all three time-points in each patient suggested that AML relapse after bone marrow transplantation (BMT) may result from the aggressive and exclusive expansion of the oncogenic cells which carry tumor-suppressor gene and/or oncogene mutation(s) and are associated with loss of donor chimerism

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Results

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