Fully automated fluorescent in situ hybridization (FISH) staining and digital analysis of HER2 in breast cancer: a validation study

Abstract

HER2 assessment is routinely used to select patients with invasive breast cancer that might benefit from HER2-targeted therapy. The aim of this study was to validate a fully automated in situ hybridization (ISH) procedure that combines the automated Leica HER2 fluorescent ISH system for Bond with supervised automated analysis with the Visia imaging D-Sight digital imaging platform. HER2 assessment was performed on 328 formalin-fixed/paraffin-embedded invasive breast cancer tumors on tissue microarrays (TMA) and 100 (50 selected IHC 2+ and 50 random IHC scores) full-sized slides of resections/biopsies obtained for diagnostic purposes previously. For digital analysis slides were pre-screened at 20x and 100x magnification for all fluorescent signals and supervised-automated scoring was performed on at least two pictures (in total at least 20 nuclei were counted) with the D-Sight HER2 FISH analysis module by two observers independently. Results were compared to data obtained previously with the manual Abbott FISH test. The overall agreement with Abbott FISH data among TMA samples and 50 selected IHC 2+ cases was 98.8% (κ = 0.94) and 93.8% (κ = 0.88), respectively. The results of 50 additionally tested unselected IHC cases were concordant with previously obtained IHC and/or FISH data. The combination of the Leica FISH system with the D-Sight digital imaging platform is a feasible method for HER2 assessment in routine clinical practice for patients with invasive breast cancer.

Fig 1. The design of the D-Sight HER2 FISH analysis module (software version 2012 2.1.2).

“Pictures”: at least 4 areas of interest are selected for analysis and pictures are taken automatically. Captured images from different focus planes are combined generating a picture with all signals clearly detectable after removal of unspecific background. “Objectives and tools”: automated nucleus selection and spot counting of the red (HER2) and the green (chromosoom 17 = CEP17) signal is evaluated and corrected by the observer if needed. “Analysis”: supervised-automated scoring is performed on at least two pictures and the ratio between HER2 and CEP17 was generated.

Fig 2. The appearance of Leica FISH HER2 staining.

Representative pictures of HER2 non-amplified invasive breast cancer specimens are shown in A) with a HER2 (red signals) to chromosoom 17 (= CEP17; green signals) ratio <2 and C) with supervised-automated nuclei and spot detection. Examples of HER2 amplified invasive breast cancer specimens are depicted in B) with a HER2/CEP17 ratio >2 and D) with supervised-automated nucleus and spot detection. DAPI counterstaining and original magnification with 100x objective.