Abstract
Detecting, characterizing, and monitoring rare populations of cells can increase testing sensitivity, give insight into disease mechanism, and inform clinical decision making. One area that can benefit from increased resolution is management of cancers in clinical remission but with measurable residual disease (MRD) by multicolor FACS. Detecting and monitoring genomic clonal resistance to treatment in the setting of MRD is technically difficult and resource intensive due to the limited amounts of disease cells. Here, we describe limited-cell FACS sequencing (LC-FACSeq), a reproducible, highly sensitive method of characterizing clonal evolution in rare cells relevant to different types of acute and chronic leukemias. We demonstrate the utility of LC-FACSeq for broad multigene gene panels and its application for monitoring sequential acquisition of mutations conferring therapy resistance and clonal evolution in long-term ibrutinib treatment of patients with chronic lymphocytic leukemia. This technique is generalizable for monitoring of other blood and marrow infiltrating cancers.
Highlights
In many types of cancer, disease relapse manifests with distinct genomic patterns of resistance and evolution
Relapse prediction and clinical decisions hinge on the ability to monitor clonal evolution in the setting of measurable residual disease (MRD) — small and persistent populations of leukemia cells whose presence directly correlates with disease progression and increased mortality
The most common approaches for monitoring clonal evolution in the setting of MRD in leukemia are surface phenotyping by multiparameter flow cytometry (MPFC), allele-specific oligonucleotide quantitative PCR (ASO-PCR), droplet digital PCR, and next-generation sequencing (NGS)
Summary
In many types of cancer, disease relapse manifests with distinct genomic patterns of resistance and evolution. Available clinical DNA sequencing and MRD monitoring techniques lack the ability to characterize emergence of clonal evolution in rare tumor cells. To address this problem, we developed a highly sensitive, fast, and cost-effective technique to characterize rare residual tumor cells in the blood or marrow. The most common approaches for monitoring clonal evolution in the setting of MRD in leukemia are surface phenotyping by multiparameter flow cytometry (MPFC), allele-specific oligonucleotide quantitative PCR (ASO-PCR), droplet digital PCR (ddPCR), and next-generation sequencing (NGS). Quantitative, and accessible, MPFC requires the analysis of millions of events to detect disease burden of 1 × 10–6 and is unable to characterize emerging clonal evolution of leukemia cells [1]. We present an assay that enables the use of limited cells acquired by FACS in tandem with targeted amplicon-based sequencing (LC-FACSeq), permitting the fast and reliable detection of more than 40 variants from as few as 50–300 sorted tumor cells by exclusion of a commonly applied step of nucleic acid extraction and purification that typically results in loss of genetic material
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