LC-FACSeq is a method for detecting rare clones in leukemia
Eileen Y. Hu, James S. Blachly, Caner Saygin, Hatice G. Ozer, Stephanie E. Workman, Arletta Lozanski, Tzyy-Jye Doong, Chi-Ling Chiang, Seema Bhat, Kerry A. Rogers, Jennifer A. Woyach, Kevin R. Coombes, Daniel Jones, Natarajan Muthusamy, Gerard Lozanski, John C. Byrd
Eileen Y. Hu, James S. Blachly, Caner Saygin, Hatice G. Ozer, Stephanie E. Workman, Arletta Lozanski, Tzyy-Jye Doong, Chi-Ling Chiang, Seema Bhat, Kerry A. Rogers, Jennifer A. Woyach, Kevin R. Coombes, Daniel Jones, Natarajan Muthusamy, Gerard Lozanski, John C. Byrd
View: Text | PDF
Resource and Technical Advance Genetics

LC-FACSeq is a method for detecting rare clones in leukemia

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.

Authors

Eileen Y. Hu, James S. Blachly, Caner Saygin, Hatice G. Ozer, Stephanie E. Workman, Arletta Lozanski, Tzyy-Jye Doong, Chi-Ling Chiang, Seema Bhat, Kerry A. Rogers, Jennifer A. Woyach, Kevin R. Coombes, Daniel Jones, Natarajan Muthusamy, Gerard Lozanski, John C. Byrd

×

Figure 2

LC-FACSeq is reproducible down to 300 cells and can detect subclones at 2% frequency.

Options: View larger image (or click on image) Download as PowerPoint
LC-FACSeq is reproducible down to 300 cells and can detect subclones at ...
(A) Poisson/β-binomial modeling of theoretical variability in VAF estimates for 50 to 500 cells for a homozygous variant sequenced with a read depth of 1000 with true VAF less than 10%. (B) Violin plots show deviations between allele frequencies of matched genetic variants called from cell dilutions and their corresponding value in bulk samples for CLL (n = 5) and AML (n = 8). Median percentage deviation from bulk and interquartile range (25th percentile, 75th percentile) for each cell titration is as follows: CLL, 500 (–0.6%, [–3.9, 0.08]); CLL, 300 (0%, [–0.8, 0.1]); CLL, 100 cells (0%, [–1.5, 0.1]); CLL, 50 (0%, [–0.8, 0.1]); AML, 500 (0%, [–2.6, 0.1]); AML, 300 (–0.2%, [–3.5, 0.1]); AML, 200 (0%, [–2.0, 0]). (C) LC-FACSeq detection of different percentages of BTK p.C481S primary B cells (0%, 2%, 5%, 8%, 10%, 25%, 50%, 75%, and 100% of 300 cells total) sorted into BTK WT primary B cells in 3 independent experiments. Mean VAF and standard deviation are as follows: (BTK p.C481S true VAF: observed VAF mean ± SD) 0%: 0.43% ± 0.8%; 2%: 2.5% ± 0.9%; 5%: 6.4% ± 4.7%; 8%: 9.0% ± 3.3%; 10%: 8.7% ± 3.4%; 25%: 24.6% ± 7.3%; 50%: 47.4% ± 4.2%; 75%: 71.7% ± 2.1%; 100%: 99.5% ± 0.8%. LC-FACSeq, limited-cell FACS sequencing; CLL, chronic lymphocytic leukemia; AML. acute myeloid leukemia; VAF, variant allele frequency.