Abstract
Intra-tumor heterogeneity is a pervasive property of human cancers that poses a major clinical challenge. Here, we describe the characterization, at the transcriptional level, of the intra-tumor topography of two prominent breast cancer biomarkers and drug targets, epidermal growth factor receptor 2 (HER2) and estrogen receptor 1 (ER) in 49 archival breast cancer samples. We developed a protocol for single-molecule RNA FISH in formalin-fixed, paraffin-embedded tissue sections (FFPE-smFISH), which enabled us to simultaneously detect and perform absolute quantification of HER2 and ER mature transcripts in single cells and multiple tumor regions. We benchmarked our method with standard diagnostic techniques, demonstrating that FFPE-smFISH is able to correctly classify breast cancers into well-established molecular subgroups. By counting transcripts in thousands of single cells, we identified different expression modes and levels of inter-cellular variability. In samples expressing both HER2 and ER, many cells co-expressed both genes, although expression levels were typically uncorrelated. Finally, we applied diversity metrics from the field of ecology to assess the intra-tumor topography of HER2 and ER gene expression, revealing that the spatial distribution of these key biomarkers can vary substantially even among breast cancers of the same subtype. Our results demonstrate that FFPE-smFISH is a reliable diagnostic assay and a powerful method for quantification of intra-tumor transcriptional heterogeneity of selected biomarkers in clinical samples.
Highlights
Intra-tumor heterogeneity (ITH) is a hallmark of human cancers that manifests itself at the genetic, epigenetic, and phenotypic level [1]
In singlemolecule RNA FISH (smFISH), individual transcripts are targeted by probes consisting of pools of 20 nucleotides-long complementary oligonucleotides, each conjugated to a single fluorophore, and are detected under a wide-field epifluorescence microscope as bright diffraction-limited spots that can be precisely enumerated and localized inside single cells (Figure 1A)
To further confirm that HER2 and estrogen receptor 1 (ER) smFISH signals overlap with tumor regions, we applied to selected cases a new pipeline, which we recently developed in our lab that enables registration of smFISH images acquired at high magnification (100X) onto a large-field scan of the same tissue section stained with H&E after smFISH, and imaged at low magnification (10X)
Summary
Intra-tumor heterogeneity (ITH) is a hallmark of human cancers that manifests itself at the genetic, epigenetic, and phenotypic level [1]. ITH of actionable mutations and more generally of any type of molecular biomarker is a major challenge, as the fraction of tumor cells expressing the www.impactjournals.com/oncotarget
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
