Liquid Biopsy Workflow Enables Single-Cell Telomere Profiling of Myeloma CTCs from Blood

Original abstract

Minimal residual disease (MRD) is a key prognostic marker for progression-free and overall survival in multiple myeloma (MM). Existing high sensitivity assays primarily focus on tumor burden assessment, rely on bone marrow sampling, and are limited in their ability to support frequent longitudinal disease monitoring. Here, we describe a proof-of-principle workflow for isolating morphologically preserved circulating tumor cells (CTCs) from peripheral blood (PB) using size-based filtration. Based on controlled spiking experiments with RPMI 8226 myeloma cells, we demonstrate an analytical limit of detection of approximately 1 tumor cell per 107 white blood cells. Isolated cells retain nuclear integrity and cytomorphology, allowing for downstream immuno-phenotyping, three-dimensional (3D) telomere fluorescence in situ hybridization (FISH), and single-cell telomere profiling, a known marker of genomic instability and disease progression in multiple myeloma. The proposed workflow demonstrated its feasibility for isolating, profiling, and analyzing plasma cells from PB of MM patients at different disease stages. It revealed distinct nuclear and telomeric features in MM CTCs compared with normal lymphocytes. The established technically robust liquid biopsy workflow enables 3D telomere profiling of MM CTCs that can be adopted for noninvasive MRD monitoring based on genomic instability rather than on the enumeration of MM plasma cells alone.Article HighlightsCurrent high-sensitivity assays for assessing minimal residual disease (MRD) in multiple myeloma (MM) patients rely on invasive bone marrow sampling and are limited by sampling bias and poor suitability for frequent longitudinal monitoring.This study presents a proof-of-principle liquid biopsy workflow that enables isolation of morphologically intact circulating tumor cells (CTCs) from peripheral blood (PB) using size-based filtration with the ScreenCell® device.Controlled spiking experiments with RPMI 8226 myeloma cells established an analytical limit of detection of approximately 1 tumor cell per 107 white blood cells.Technical feasibility of the new workflow for isolating intact CTCs from liquid biopsy was confirmed in a cohort of 20 newly diagnosed MM patients at diagnosis, during induction therapy, and after relapse, supporting its potential utility for longitudinal disease monitoring.Isolated CTCs were successfully immunophenotyped and subjected to quantitative three-dimensional telomere fluorescence in situ hybridization (FISH), allowing single-cell analysis of telomere length, number, aggregation, nuclear volume, and spatial distribution.Quantitative telomere profiling revealed statistically significant differences in nuclear and telomeric parameters between MM CTCs and normal lymphocytes, consistent with known markers of genomic instability and disease aggressiveness in MM.By combining enumeration with risk assessment based on telomere profiling, the current workflow can provide clinicians with much-needed biological insight beyond mere tumor burden assessment. Incorporating minimally invasive telomere profile-based risk assessment into MRD guidelines may guide treatment decisions in cases of sustained MRD and inform the need for new treatment regimens when residual disease is detected.