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

Westernized high-fat diet accelerates weight loss in dextran sulfate sodium-induced colitis in mice, which is further aggravated by supplementation of heme

<p>The Western diet, rich in fat and red meat, predisposes for inflammatory bowel disease (IBD); however, little is known about mechanisms involved. Red meat contains high levels of heme, a well-known inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1). Pharmacological induction of HO-1 ameliorates experimental colitis. We analyzed the effect of a westernized high-fat (HF) diet supplemented with heme on intestinal HO-1 expression and dextran sulfate sodium (DSS)-induced colitis.</p><p>Mice were fed chow or HF diets for 2 weeks. In the second week, the HF diet was supplemented with or without 0.5 mu mol/g heme. Subsequently, the 3 diet groups were given drinking water with or without 4% DSS to induce colitis.</p><p>Significant body weight reduction was first observed after 4 days in the chow/DSS mice (-5 +/- 3%), whereas this was evident already after 2 days (-6 +/- 2%) in HF/DSS mice, showing increased weight loss compared to chow/DSS mice in the following days. Heme supplementation further aggravated DSS-induced weight loss in HF mice (-18 +/- 4% vs. -7 +/- 5% for HF+heme/DSS vs. HF/DSS, P</p><p>A westernized diet accelerates DSS-induced weight loss in mice, which is further aggravated by heme, despite the induction of HO-1 in the colon epithelium. Our data warrant a detailed analysis of the association of (red) meat-containing diets and the development of IBD. (C) 2013 Elsevier Inc. All rights reserved.</p>

Effects of active and passive smoking on disease course of Crohn's disease and ulcerative colitis

Background: Smoking is a remarkable risk factor for inflammatory bowel disease (IBD). aggravating Crohn's disease (CD) while having beneficial effects On Ulcerative colitis (UC). We Studied the effects of active and passive smoking in Dutch IBD patients. Methods: A questionnaire focusing Oil cigarette smoke exposure was sent to 820 IBD patients. Returned questionnaries were incorporated into a retrospective chart review, containing details about disease behavior and received therapy. Results: In all, 675 IBD patients (380 [56%] CD and 295 [44%] UC) responded. At diagnosis there were 52% smokers in CD, 41% it) the general Population, and 28% in UC. The number of present smokers in CD is lower than in the general population (26% versus 35%). No detrimental effects of active smoking oil CD were observed, but passive smokers needed immunosuppressants and infliximab more frequently than nonpassive smokers. Active smoking had beneficial effects oil UC, indicated by reduced rates of colectomy, primary sclerosing cholangitis, and backwash-ileitis in active smokers compared to never smokers, and higher daily cigarette dose correlated with less extensive colitis and a lower need for therapy. Furthermore, smoking cessation after diagnosis was, detrimental for UC patients, indicated by increased needs for steroids and hospitalizations for patients that stopped smoking after compared to before the diagnosis. Conclusions: Active smoking is a risk factor for CD, but does not affect the outcome passive smoking is detrimental for the outcome of CD patients. In UC, active smoking shows dose-dependent beneficial effects. Our data suggest that passive smoking is a novel risk factor for CD

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

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

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