Fully Automated Fluorescent in situ Hybridization (FISH) Staining and Digital Analysis of HER2 in Breast Cancer:A Validation Study

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

Comparisons of Leica <i>HER2</i> FISH and DAKO HER2 IHC of 50 consecutively collected invasive breast cancer tissue specimens (resection / biopsy) from routine practice.

<p>IHC, immunohistochemistry; FISH, fluorescence <i>in situ</i> hybridization. 1x missing data because no signals were detected with Leica FISH.</p><p>^a ratio ≥ 2.2.</p><p>^b ratio = 1.8–2.2.</p><p>^c ratio <1.8.</p><p>Comparisons of Leica <i>HER2</i> FISH and DAKO HER2 IHC of 50 consecutively collected invasive breast cancer tissue specimens (resection / biopsy) from routine practice.</p

The appearance of Leica FISH <i>HER2</i> staining.

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

Comparisons of automated Leica <i>HER2</i> FISH with digital analysis and manual Abbott <i>HER2</i> FISH with manual analysis in 20 full-sized slides of tissue specimens used for TMA blocks of invasive breast cancer.

<p>FISH, fluorescence <i>in situ</i> hybridization.</p><p>^a ratio ≥ 2.2.</p><p>^b ratio = 1.8–2.2.</p><p>^c ratio <1.8.</p><p>^d between brackets equivocal ratio after additional count of 20 nuclei <2.0 or ≥2.0.</p><p>^e 3 categories (amplified / equivocal / non-amplified).</p><p>^f 2 categories (amplified ratio ≥ 2.0 / non-amplified ratio <2.0).</p><p>Comparisons of automated Leica <i>HER2</i> FISH with digital analysis and manual Abbott <i>HER2</i> FISH with manual analysis in 20 full-sized slides of tissue specimens used for TMA blocks of invasive breast cancer.</p

Comparisons of automated Leica <i>HER2</i> FISH with digital analysis and manual Abbott <i>HER2</i> FISH with manual analysis of 50 invasive breast cancer tissue specimens (resection / biopsy) with IHC 2+ scores.

<p>FISH, fluorescence <i>in situ</i> hybridization.</p><p>^a ratio ≥ 2.2.</p><p>^b ratio = 1.8–2.2.</p><p>^c ratio <1.8.</p><p>^d 3 categories (amplified / equivocal / non-amplified).</p><p>^e 2x) reanalysis Leica FISH confirms amplification Abbott FISH; 1x) new staining Abbott FISH confirms non-amplified score Leica FISH (first Abbott FISH contained 3 biopsies, whereas the biopsy in the middle is missing in the new Abbott FISH and Leica FISH). 2x missing data because no signals were detected with Leica FISH.</p><p>Comparisons of automated Leica <i>HER2</i> FISH with digital analysis and manual Abbott <i>HER2</i> FISH with manual analysis of 50 invasive breast cancer tissue specimens (resection / biopsy) with IHC 2+ scores.</p

Comparisons of automated Leica <i>HER2</i> FISH with digital analysis and manual Abbott <i>HER2</i> FISH with manual analysis in tissue micro arrays (TMAs) including 328 invasive breast cancer tissue specimens.

<p>FISH, fluorescence <i>in situ</i> hybridization.</p><p>^a ratio ≥ 2.2.</p><p>^b ratio = 1.8–2.2.</p><p>^c ratio <1.8.</p><p>^d between brackets equivocal ratio after additional count of 20 nuclei <2.0 or ≥2.0.</p><p>^e 3 categories (amplified / equivocal / non-amplified).</p><p>^f 2 categories (amplified ratio ≥ 2.0 / non-amplified ratio <2.0).</p><p>^g Bold characters indicate discordant results.</p><p>Comparisons of automated Leica <i>HER2</i> FISH with digital analysis and manual Abbott <i>HER2</i> FISH with manual analysis in tissue micro arrays (TMAs) including 328 invasive breast cancer tissue specimens.</p