ESR1 and neoplasm: To further confirm that HER2 and ER smFISH signals overlap with tumor regions, we applied to selected cases a new pipeline, which we recently developed in our lab (unpublished data) that enables registration of smFISH images acquired at high magnification (100X) onto a large-field (up to 2 × 2 cm) scan of the same tissue section stained with H&E after smFISH, and imaged at low magnification (10X) (http://tumorheterogeneity.eu/ and Materials and Methods).