Clonal Architecture of Secondary Acute Myeloid Leukemia Defined by Single-Cell Sequencing

Abstract

Next-generation sequencing has been used to infer the clonality of heterogeneous tumor samples. These analyses yield specific predictions—the population frequency of individual clones, their genetic composition, and their evolutionary relationships—which we set out to test by sequencing individual cells from three subjects diagnosed with secondary acute myeloid leukemia, each of whom had been previously characterized by whole genome sequencing of unfractionated tumor samples. Single-cell mutation profiling strongly supported the clonal architecture implied by the analysis of bulk material. In addition, it resolved the clonal assignment of single nucleotide variants that had been initially ambiguous and identified areas of previously unappreciated complexity. Accordingly, we find that many of the key assumptions underlying the analysis of tumor clonality by deep sequencing of unfractionated material are valid. Furthermore, we illustrate a single-cell sequencing strategy for interrogating the clonal relationships among known variants that is cost-effective, scalable, and adaptable to the analysis of both hematopoietic and solid tumors, or any heterogeneous population of cells.

Dissecting the clonal origins of childhood acute lymphoblastic leukemia by single-cell genomics

“…The ADO rate in the primary tumor cells was modestly higher than the median ADO rate of 15.6% observed in a control lymphoblastoid cell line and consistent with previous reports showing higher ADO rates in primary patient samples (12). These rates are also consistent with other single-cell primary sample cancer sequencing approaches using MDA and lower than approaches that have used PCR-based genome amplification (12)(13)(14)(15). The ADO data and percent of cells removed from further analyses as a result of these quality control measures are summarized in SI Appendix, modeling-based approach where we first execute an expectation maximization algorithm on a multivariate Bernoulli model (SI Appendix, Fig.…”

“…In addition, two papers established the feasibility of using isothermal single-cell WGA to detect SNVs in cancer samples, although neither study was able to determine clonal structures that were shown to represent the bulk samples (12,13). Another recent study was unable to determine the clonal structure of myeloid leukemia samples with single-cell data alone, as they evaluated 12 cells per patient and lost an average of 55% of the genomes of those cells during WGA and uneven sequencing after target capture (15). Two other studies used single-cell sorting followed by target-specific preamplification to perform quantitative PCR (qPCR)-based CNV analyses, as well as allele-discriminating qPCR-based SNV detection (8,27).…”

Acute promyelocytic leukemia co-existing with JAK2 V617F positive myeloproliferative neoplasm: a case report

“…JAK-2 inhibition by ruxolitinib in the in vitro studies on NB-4 APL cells treated with ATRA did not affect CLL-2 and IL-8 levels, supporting the importance of JAK2 in downstream activation as previously described (1,2). However, the role of JAK-2 inhibition in the management of differentiation syndrome has not been (23)(24)(25). Since splenomegaly and FLT3-TKD were also present at the time of diagnosis, we hypothesize that the JAK2 V617F mutated MPN clone (likely PMF) was present first and that additional mutations like FLT3 and PML/RARa took place later and then led to the development of APL (26)(27)(28)(29)(30)(31)(32).…”

Clinical Implications of Novel Genomic Discoveries in Chronic Lymphocytic Leukemia

“…76 Single-cell WGS approaches can potentially provide a comprehensive snapshot of the subclonal composition of a population, 77,78 but this is not high throughput nor cost effective at the present time. Droplet digital polymerase chain reaction technology is an alternative method for analyzing single cells by compartmentalizing them using water-oil emulsion at high throughputs.…”