RAS/RAF landscape in monoclonal plasma cell conditions.

ABSTRACT: Multiple myeloma is characterized by a huge heterogeneity at the molecular level. The RAS/RAF pathway is the most frequently mutated, in ∼50% of the patients. However, these mutations are frequently subclonal, suggesting a secondary event. Because these genes are part of our routine next-generation sequencing panel, we analyzed >10 000 patients with different plasma cell disorders to describe the RAS/RAF landscape. In this large cohort of patients, almost 61% of the patients presented a RAS/RAF mutation at diagnosis or relapse, but much lower frequencies occurred in presymptomatic cases. Of note, the mutations were different from that observed in solid tumors (higher proportions of Q61 mutations). In 29 patients with 2 different mutations, we were able to perform single-cell sequencing, showing that in most cases, mutations occurred in different subclones, suggesting an ongoing mutational process. These findings suggest that the RAS/RAF pathway is not an attractive target, both on therapeutic and residual disease assessment points of view.

High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics.

To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cancer cells confined to droplets. The barcodes are then used to reassemble the genetic profiles of cells from next-generation sequencing data. By using this approach, we sequenced longitudinally collected acute myeloid leukemia (AML) tumor populations from two patients and genotyped up to 62 disease relevant loci across more than 16,000 individual cells. Targeted single-cell sequencing was able to sensitively identify cells harboring pathogenic mutations during complete remission and uncovered complex clonal evolution within AML tumors that was not observable with bulk sequencing. We anticipate that this approach will make feasible the routine analysis of AML heterogeneity, leading to improved stratification and therapy selection for the disease.

Microfluidic droplet enrichment for targeted sequencing.

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications.

RNA-Seq following PCR-based sorting reveals rare cell transcriptional signatures.

BACKGROUND: Rare cell subtypes can profoundly impact the course of human health and disease, yet their presence within a sample is often missed with bulk molecular analysis. Single-cell analysis tools such as FACS, FISH-FC and single-cell barcode-based sequencing can investigate cellular heterogeneity; however, they have significant limitations that impede their ability to identify and transcriptionally characterize many rare cell subpopulations. RESULTS: PCR-activated cell sorting (PACS) is a novel cytometry method that uses single-cell TaqMan PCR reactions performed in microfluidic droplets to identify and isolate cell subtypes with high-throughput. Here, we extend this method and demonstrate that PACS enables high-dimensional molecular profiling on TaqMan-targeted cells. Using a random priming RNA-Seq strategy, we obtained high-fidelity transcriptome measurements following PACS sorting of prostate cancer cells from a heterogeneous population. The sequencing data revealed prostate cancer gene expression profiles that were obscured in the unsorted populations. Single-cell expression analysis with PACS was subsequently used to confirm a number of the differentially expressed genes identified with RNA sequencing. CONCLUSIONS: PACS requires minimal sample processing, uses readily available TaqMan assays and can isolate cell subtypes with high sensitivity. We have now validated a method for performing next-generation sequencing on mRNA obtained from PACS isolated cells. This capability makes PACS well suited for transcriptional profiling of rare cells from complex populations to obtain maximal biological insight into cell states and behaviors.

Identification and genetic analysis of cancer cells with PCR-activated cell sorting.

Cell sorting is a central tool in life science research for analyzing cellular heterogeneity or enriching rare cells out of large populations. Although methods like FACS and FISH-FC can characterize and isolate cells from heterogeneous populations, they are limited by their reliance on antibodies, or the requirement to chemically fix cells. We introduce a new cell sorting technology that robustly sorts based on sequence-specific analysis of cellular nucleic acids. Our approach, PCR-activated cell sorting (PACS), uses TaqMan PCR to detect nucleic acids within single cells and trigger their sorting. With this method, we identified and sorted prostate cancer cells from a heterogeneous population by performing >132 000 simultaneous single-cell TaqMan RT-PCR reactions targeting vimentin mRNA. Following vimentin-positive droplet sorting and downstream analysis of recovered nucleic acids, we found that cancer-specific genomes and transcripts were significantly enriched. Additionally, we demonstrate that PACS can be used to sort and enrich cells via TaqMan PCR reactions targeting single-copy genomic DNA. PACS provides a general new technical capability that expands the application space of cell sorting by enabling sorting based on cellular information not amenable to existing approaches.

Ultrahigh-throughput Mammalian single-cell reverse-transcriptase polymerase chain reaction in microfluidic drops.

The behaviors of complex biological systems are often dictated by the properties of their heterogeneous and sometimes rare cellular constituents. Correspondingly, the analysis of individual cells from a heterogeneous population can reveal information not obtainable by ensemble measurements. Reverse-transcriptase polymerase chain reaction (RT-PCR) is a widely used method that enables transcriptional profiling and sequencing analysis on bulk populations of cells. Major barriers to successfully implementing this technique for mammalian single-cell studies are the labor, cost, and low-throughput associated with current approaches. In this report, we describe a novel droplet-based microfluidic system for performing ~50000 single-cell RT-PCR reactions in a single experiment while consuming a minimal amount of reagent. Using cell type-specific staining and TaqMan RT-PCR probes, we demonstrate the identification of specific cells from a mixed human cell population. The throughput, robust detection rate and specificity of this method makes it well-suited for characterizing large, heterogeneous populations of cells at the transcriptional level.

Single-cell genotypic and phenotypic analysis of measurable residual disease in acute myeloid leukemia.

Measurable residual disease (MRD), defined as the population of cancer cells that persist following therapy, serves as the critical reservoir for disease relapse in acute myeloid leukemia and other malignancies. Understanding the biology enabling MRD clones to resist therapy is necessary to guide the development of more effective curative treatments. Discriminating between residual leukemic clones, preleukemic clones, and normal precursors remains a challenge with current MRD tools. Here, we developed a single-cell MRD (scMRD) assay by combining flow cytometric enrichment of the targeted precursor/blast population with integrated single-cell DNA sequencing and immunophenotyping. Our scMRD assay shows high sensitivity of approximately 0.01%, deconvolutes clonal architecture, and provides clone-specific immunophenotypic data. In summary, our scMRD assay enhances MRD detection and simultaneously illuminates the clonal architecture of clonal hematopoiesis/preleukemic and leukemic cells surviving acute myeloid leukemia therapy.

Personalized Single-Cell Proteogenomics to Distinguish Acute Myeloid Leukemia from Non-Malignant Clonal Hematopoiesis.

Genetic mutations associated with acute myeloid leukemia (AML) also occur in age-related clonal hematopoiesis, often in the same individual. This makes confident assignment of detected variants to malignancy challenging. The issue is particularly crucial for AML post-treatment measurable residual disease monitoring, where results can be discordant between genetic sequencing and flow cytometry. We show here, that it is possible to distinguish AML from clonal hematopoiesis and to resolve the immunophenotypic identity of clonal architecture. To achieve this, we first design patient-specific DNA probes based on patient's whole-genome sequencing, and then use them for patient-personalized single-cell DNA sequencing with simultaneous single-cell antibody-oligonucleotide sequencing. Examples illustrate AML arising from DNMT3A and TET2 mutated clones as well as independently. The ability to personalize single-cell proteogenomic assessment for individual patients based on leukemia-specific genomic features has implications for ongoing AML precision medicine efforts.

Lgr6 is a stem cell marker in mouse skin squamous cell carcinoma.

The G-protein-coupled receptors LGR4, LGR5 and LGR6 are Wnt signaling mediators, but their functions in squamous cell carcinoma (SCC) are unclear. Using lineage tracing in Lgr5-EGFP-CreERT2/Rosa26-Tomato and Lgr6-EGFP-CreERT2/Rosa26-Tomato reporter mice, we demonstrate that Lgr6, but not Lgr5, acts as an epithelial stem cell marker in SCCs in vivo. We identify, by single-molecule in situ hybridization and cell sorting, rare cells positive for Lgr6 expression in immortalized keratinocytes and show that their frequency increases in advanced SCCs. Lgr6 expression is enriched in cells with stem cell characteristics, and Lgr6 downregulation in vivo causes increased epidermal proliferation with expanded lineage tracing from epidermal stem cells positive for Lgr6 expression. Surprisingly, mice with germline knockout of Lgr6 are predisposed to SCC development, through a mechanism that includes compensatory upregulation of Lgr5. These data provide a model for human patients with germline loss-of-function mutations in Wnt pathway genes, including RSPO1 or LGR4, who show increased susceptibility to squamous tumor development.

Accurate microfluidic sorting of droplets at 30 kHz.

Fluorescence-activated droplet sorting is an important tool for droplet microfluidic workflows, but published approaches are unable to surpass throughputs of a few kilohertz. We present a new geometry that replaces the hard divider separating the outlets with a gapped divider, allowing sorting over ten times faster.

Generating electric fields in PDMS microfluidic devices with salt water electrodes.

Droplet merging and sorting in microfluidic devices usually rely on electric fields generated by solid metal electrodes. We show that simpler and more reliable salt water electrodes, despite their lower conductivity, can perform the same droplet manipulations at the same voltages.

Picoinjection of microfluidic drops without metal electrodes.

Existing methods for picoinjecting reagents into microfluidic drops require metal electrodes integrated into the microfluidic chip. The integration of these electrodes adds cumbersome and error-prone steps to the device fabrication process. We have developed a technique that obviates the needs for metal electrodes during picoinjection. Instead, it uses the injection fluid itself as an electrode, since most biological reagents contain dissolved electrolytes and are conductive. By eliminating the electrodes, we reduce device fabrication time and complexity, and make the devices more robust. In addition, with our approach, the injection volume depends on the voltage applied to the picoinjection solution; this allows us to rapidly adjust the volume injected by modulating the applied voltage. We demonstrate that our technique is compatible with reagents incorporating common biological compounds, including buffers, enzymes, and nucleic acids.

Adding reagent to droplets with controlled rupture of encapsulated double emulsions.

We present a method to add reagent to microfluidic droplets by enveloping them as a double emulsions in reagent-filled droplets and then rupturing them with an electric field. When the double emulsions rupture, they release their contents into the enveloping droplets, ensuring mixing with reagent while limiting cross-droplet contamination.

Picoinjection enables digital detection of RNA with droplet rt-PCR.

The ability to add reagents to drops in a sequential fashion is necessary for numerous applications of microfluidics in biology. An important method for accomplishing this is picoinjection, a technique in which reagents are injected into aqueous drops using an electric field. While picoinjection has been shown to allow the precise addition of reagents to drops, its compatibility with biological reactions is yet to be thoroughly demonstrated. Here, we investigate the compatibility of picoinjection with digital RT-PCR Taqman assays, reactions that incorporate nucleic acids, enzymes, and other common biological reagents. We find that picoinjection is compatible with this assay and enables the detection of RNA transcripts at rates comparable to workflows not incorporating picoinjection. We also find that picoinjection results in negligible transfer of material between drops and that the drops faithfully retain their compartmentalization.