Quantification of HER2 and estrogen receptor heterogeneity in breast cancer by single-molecule RNA fluorescence in situ hybridization

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.

Synthetic lethality between androgen receptor signalling and the PARP pathway in prostate cancer

“…To quantify the smFISH signal, we selected the 5 most in-focus images in each stack and then counted all the mRNA molecules in each field of view using custom scripts in MATLAB ® , as previously described 29 . To get an estimate of the mRNA density per cell (dots per µm 3 ), we used an approach similar to what we recently described for quantifying HER2 and estrogen receptor transcriptional heterogeneity in breast cancer 30 : we split the z-projection of the mRNA dots identified in each stack into a regular grid of 13 × 13 squared pseudo-cells, and then divided the number of dots in each pseudo-cell by the number of focal planes minus one times the distance between each plane times the pseudo-cell area. In order to exclude possible background dots, we only considered pseudo-cells containing ≥3 mRNA dots.…”

Spatially Resolved Transcriptomes—Next Generation Tools for Tissue Exploration

“…This method is superior for both RNA quantification and spatial resolution compared to the earlier cryosectioning protocol. [8] However, transcriptome-wide maps using tomo-seq can only be www.advancedsciencenews.com www.bioessays-journal.com Fresh-frozen or FFPE [57] Subcellular Targeted Nearly 100% [58] Fresh-frozen Subcellular Targeted 84% [19] + Highly multiplex − Need specialized equipment Cost becomes significant for increased number of targets Limited field of view Yes, in collaboration with inventors MERFISH Fresh-frozen Subcellular Targeted 80% [21] + Highly multiplex − Need specialized equipment Cost becomes significant for increased number of targets Limited field of view Yes seqFISH+ Fresh-frozen Subcellular Targeted 49% [24] + Exceedingly high multiplex − Cost becomes significant for increased number of targetsLimited field of view No osmFISH Fresh-frozen Subcellular Targeted Nearly 100%, but with 5.3 ± 6.9% RNA losses per cycle [25] + Fresh-frozen and FFPE [59] Subcellular Targeted 30% [60] + Subcellular resolutionAbility to detect SNVs − A priori chosen targets of limited quantity Yes, commercialized spin-off…”

Spatial Deconvolution of HER2-positive Breast Tumors Reveals Novel Intercellular Relationships

“…Interpatient heterogeneity can be expected when working with tumor samples. [24] Thus, to properly capture the nuances of each patient's molecular profile and not risk quenching weak signals, we analyzed each patient separately, then compared the outcomes.…”