title: FuseFISH: robust detection of transcribed gene fusions in single cells.
abstract: Transcribed gene fusions are key biomarkers in many hematologic and solid tumors, often representing the primary oncogenic driver mutation. Here, we report an experimental and computational pipeline for detecting fusion transcripts using single-molecule RNA FISH and unbiased correlation analysis (FuseFISH). We constructed a genome-wide database of optimal oligonucleotide sequences, enabling quick design of FuseFISH probes against known and novel fusions. We implemented FuseFISH in cell lines, tissue sections, and purified RNA, reliably detecting one BCR-ABL1 positive in 10,000 negative cells. In 34 hematologic samples, we detected BCR-ABL1 transcripts with high specificity and sensitivity. Finally, we measured BCR-ABL1 expression heterogeneity and dynamics in single CML cells exposed to the kinase inhibitor Nilotinib. Our resource and methods are ideal for streamlined validation of fusions newly identified by next-generation sequencing, and they pave the way to studying the impact of fusion expression variability on clinical outcome.
authors.affiliation: Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Stefan
authors.initials: S
authors.lastname: Semrau
authors.affiliation: Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Nicola
authors.initials: N
authors.lastname: Crosetto
authors.affiliation: Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Magda
authors.initials: M
authors.lastname: Bienko
authors.affiliation: Division of Hematology, Fondazione IRCCS Policlinico San Matteo, University of Pavia, 27100 Pavia, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Boni
authors.affiliation: Division of Hematology, Fondazione IRCCS Policlinico San Matteo, University of Pavia, 27100 Pavia, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Bernasconi
authors.affiliation: Department of Pathology, Children's Hospital, Boston, MA 02138, USA; Harvard Medical School, Boston, MA 02138, USA; Department of Molecular Biotechnology and Health Sciences, University of Torino, 10126 Torino, Italy. Electronic address: roberto.chiarle@childrens.harvard.edu.
authors.forename: Roberto
authors.initials: R
authors.lastname: Chiarle
authors.affiliation: Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical Center Utrecht, 3584 CT Utrecht, The Netherlands. Electronic address: a.vanoudenaarden@hubrecht.eu.
authors.forename: Alexander
authors.initials: A
authors.lastname: van Oudenaarden
mesh_terms: D045744:Cell Line, Tumor; D019295:Computational Biology / Q000379:methods; D030561:Databases, Nucleic Acid; D016044:Fusion Proteins, bcr-abl / Q000235:genetics / Q000378:metabolism*; D015894:Genome, Human; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: C581634:BCR-ABL1 fusion protein, human; D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl