BACKGROUND:Positive HER2 status identifies breast carcinomas that might respond to trastuzumab treatment. Manual HER2 fluorescent in situ hybridisation (FISH) is the most readily used method to detect HER2 gene amplification which defines positive HER2 status in addition to HER2 protein overexpression. Automation of HER2 FISH may improve HER2 gene testing. The aim of our study was to evaluate an automated HER2 FISH assay for assessing the HER2 genomic status.METHODS:Core biopsies of 100 invasive breast carcinomas were analysed in parallel using the PathVysion™ HER-2 DNA Probe Kit and the Leica HER2 FISH System for BOND™. To assess inter-method agreement, concordance analysis was performed for various numerical and categorical HER2/CEP17 FISH parameters.RESULTS:Carcinomas with all HER2 immunohistochemical scores were included (0+: 20; 1+: 20; 2+: 30; 3+: 30). Using either HER2/CEP17 ratio >2.2 or ≥2.0 as criterion for HER2 amplification, high levels of concordance were observed between automated and manual FISH (concordance rate 96%, κ coefficient 0.92). High levels of inter-method agreement were also found for HER2 copy number, CEP17 copy number, HER2/CEP17 ratio, the percentage of carcinoma cells with HER2/CEP17 ratio >2.2, and the presence of HER2 genetic heterogeneity, HER2 clusters and CEP17 polyploidy.CONCLUSIONS:HER2 testing using automated FISH is feasible on breast carcinoma core biopsies. Automated HER2 FISH using the Leica HER2 FISH System for BOND is a practical and efficient alternative to manual HER2 FISH in evaluating the HER2 status of primary invasive breast carcinomas.

Aims To determine the frequency of HER2 genetic heterogeneity according to the recent American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) definition (2009) in invasive breast carcinoma, and to identify clinicopathological features that characterise breast carcinomas with HER2 genetic heterogeneity. Methods 530 invasive breast carcinomas were retrospectively analysed for HER2 genetic heterogeneity, and investigated for a potential association of HER2 genetic heterogeneity with other HER2 FISH findings, clinicopathological parameters, oestrogen/progesterone receptor expression and DNA cytometric parameters in breast carcinomas with an equivocal (2+) HER2 immunohistochemical score. Results The overall frequency of HER2 genetic heterogeneity was 14.7% in a cohort of 218 consecutive breast carcinomas. HER2 genetic heterogeneity was most frequent in invasive breast carcinomas with an equivocal (2+) HER2 immunohistochemical score. Among the 151 carcinomas lacking HER2 amplification, 16.1% showed HER2 genetic heterogeneity. In an extended cohort of 345 carcinomas with a (2+) HER2 score, the frequency of HER2 genetic heterogeneity was 41%, and was associated with the absence of HER2 gene clusters, chromosome 17 polysomy, histological tumour grade, DNA ploidy category and 5c exceeding rate. Conclusion HER2 genetic heterogeneity according to the ASCO/CAP definition is frequent in breast carcinoma, and is most often present in carcinomas with an equivocal (2+) HER2 score. Many carcinomas with HER2 genetic heterogeneity have a negative HER2 amplification status, although they contain a significant number of tumour cells with HER2 gene amplification. Single cell scoring of the HER2 /17 centromeric probe (CEP17) ratio is necessary to identify carcinomas with HER2 genetic heterogeneity, because they lack specific clinicopathological characteristics.

Intratumorale Heterogenität von HER2-Proteinexpression und HER2-Genamplifikation erschweren die zuverlässige Bestimmung des HER2-Status bei einem Teil der invasiven Mammakarzinome. Häufigkeit und klinische Bedeutung der genetischen HER2-Heterogenität sind unbekannt, teils wegen Fehlens einheitlicher Kriterien zu ihrer Bestimmung. Empfehlungen von ASCO/CAP (2009) definieren genetische HER2-Heterogenität als das Vorhandensein von >5 bis <50% Tumorzellen mit einer HER2/CEP17-Ratio von >2,2. Ein von uns entwickeltes Auswerteinstrument (Excel-Arbeitsblatt) ermöglicht die einfache und zuverlässige Diagnosestellung einer genetischen HER2-Heterogenität gemäß ASCO/CAP-Kriterien. Es könnte zukünftig Einsatz in der HER2-Routinediagnostik sowie in Studien zur bisher unbekannten prädiktiven Bedeutung von genetischer HER2-Heterogenität finden.

Intratumoral heterogeneity of HER2 protein expression and HER2 gene amplification can negatively affect determination of HER2 status in a subset of invasive breast carcinomas. The frequency and clinical significance of HER2 genetic heterogeneity are unknown due to the lack of uniform criteria for diagnosis. Recent ASCO/CAP guidelines (2009) define HER2 genetic heterogeneity as the presence of between 5% and 50% of tumor cells with a HER2/CEP17 ratio >2.2. We describe a tool (a customized Excel spreadsheet) for easy and reproducible diagnosis of HER2 genetic heterogeneity according to ASCO/CAP criteria. Our tool may be useful for routine HER2 diagnostics and for studies to analyse the hitherto unknown predictive significance of HER2 genetic heterogeneity.

BACKGROUND AND STUDY AIMS The current gold standard in Barrett's esophagus monitoring consists of four-quadrant biopsies every 1-2 cm in accordance with the Seattle protocol. Adding brush cytology processed by digital image cytometry (DICM) may further increase the detection of patients with Barrett's esophagus who are at risk of neoplasia. The aim of the present study was to assess the additional diagnostic value and accuracy of DICM when added to the standard histological analysis in a cross-sectional multicenter study of patients with Barrett's esophagus in Switzerland. METHODS One hundred sixty-four patients with Barrett's esophagus underwent 239 endoscopies with biopsy and brush cytology. DICM was carried out on 239 cytology specimens. Measures of the test accuracy of DICM (relative risk, sensitivity, specificity, likelihood ratios) were obtained by dichotomizing the histopathology results (high-grade dysplasia or adenocarcinoma vs. all others) and DICM results (aneuploidy/intermediate pattern vs. diploidy). RESULTS DICM revealed diploidy in 83% of 239 endoscopies, an intermediate pattern in 8.8%, and aneuploidy in 8.4%. An intermediate DICM result carried a relative risk (RR) of 12 and aneuploidy a RR of 27 for high-grade dysplasia/adenocarcinoma. Adding DICM to the standard biopsy protocol, a pathological cytometry result (aneuploid or intermediate) was found in 25 of 239 endoscopies (11%; 18 patients) with low-risk histology (no high-grade dysplasia or adenocarcinoma). During follow-up of 14 of these 18 patients, histological deterioration was seen in 3 (21%). CONCLUSION DICM from brush cytology may add important information to a standard biopsy protocol by identifying a subgroup of BE-patients with high-risk cellular abnormalities.

Background and Aims: Flowcytometry on biopsy specimens has been previously shown to identify patients with Barrett's esophagus at high risk for progression to cancer. Patients with aneuploidy or increased 4N have a 5-yr cumulative cancer incidence of 28% (Am J Gastroenterol 2000;95:1669-1676). Using endoscopic brush cytology we studied the additional diagnostic benefit of Image Cytometry (ICM) of specialised intestinal metaplasia (SIM) compared to the histological diagnosis obtained from two separate endoscopies. Primary endpoints were detection of early cancer (EC) and high-grade dysplasia (HGD), secondary endpoint was low-grade dysplasia (LGD). Methods: 200 patients (73% male; mean age 67 yrs) with Barrett's esophagus (BE, mean segment length: 4.6 cm) were included, of whom 61% underwent two endoscopies (total of 325 examinations). In total, ICMs were obtained in 240 endoscopies from 162 patients. ICM was performed with a CCD 3-chip color videocamera Sony and AutoCyte QUIC DNA workstation on Feulgen restained cytology smears. DNA patterns were defined as diploid (2c), intermediate (diploid stemline and >10% cells below 4.5c threshhold) and aneuploid (>15% aneuploid cells or >3 cells > 9c). Results: 20 (8.3%) of 240 ICM examinations showed aneuploidy with the corresponding histological diagnosis of 3 EC (15%) , 8 HGD (40%), 5 LGD (30%), 1 indefinite for dysplasia (5%) and 3 SIM (15%) without dysplasia. 21 (8.8%) intermediate DNA-patterns at ICM were recorded at 2 endoscopies with EC, 3 endoscopies with HGD and 16 with indefinite for dysplasia. 199 (82.9%) ICMs showed diploidy in patients with EC (n = 1, 0.5%), HGD (n = 3, 1.5%), LGD (n = 10, 5%), indefinite for dysplasia (n = 20, 10%) and SIM (n = 164, 82.9%). Thus, ICM detected 25 additional high risk constellations in endoscopies with histology of LGD, indefinite for dysplasia (ID) and SIM or 1 in every 10 endoscopies with low-risk histology from modified gold standard four-quadrant biopsies. Conclusions: The data suggest that in this population of patients referred to a tertiary center, combined brush cytology and image cytometry in Barrett's esophagus identifies 1 additional high risk patients with aneuploid and intermediate DNA-pattern in every 10 endoscopies. Prospective studies need to demonstrate that this additional information affects patient management in a way ultimately improving outcomes.

Background: We have previously shown that Digital Image Cytometry (DICM) of brush cytology in Barrett's Esophagus (BE) identifies additional high risk patients with aneuploid and intermediate DNA-pattern. Similarly, flowcytometry of biopsy specimens with aneuploidy or increased 4N combined with p53 loss of heterozygosity (LOH) may identify patients at risk of progression to cancer. Methods: This prospective cohort study aimed to assess specific genetic changes of p53 by Fluorescence in situ Hybridization (FISH) as compared to DICM in cytologic specimens of patients with BE: a) no dysplasia (No D) b) low-grade dysplasia (LGD), c) high-grade dysplasia (HGD) and d) early cancer (Ca). We examined 58 brush specimens from 43 patients (27 male; mean age 64 yrs) with BE by DICM on Feulgen restained cytology smears. DNA patterns were defined as diploid, intermediate and aneuploid. Fifty cytologies (94%) were further processed by two-color FISH using a locus-specific probe for 17p13.1 (p53 gene) with an enumeration probe. A pathological FISH probe was defined as loss of p53 (LOH) in more than 6.7% of cells (> mean + 3 SD of diploid controls). Results: 15/58 (26%) of DICM examinations showed aneuploidy with p53 LOH by FISH in 10/11 (91%). 13/58 (22%) intermediate DNA-patterns were diagnosed with p53 LOH in 12/13 (92%). 30/58 (52%) brush cytologies were diploid with p53 LOH by FISH in 5/30 (17%). Thus, pathological DICM (aneuploid or intermediate pattern) correlated with p53 LOH by FISH in 23/25 (92%) of brush cytologies, yielding good agreement beyond chance (Cohen's kappa of 0.74). The combined analysis selected a high-risk constellation with p53 LOH in 21/42 (50%) of patients negative for HGD or Ca. Conclusion: Combined DICM with p53 LOH by FISH detects patients with BE without HGD or Ca who may be at increased risk for neoplastic progression according to current literature. Provided that prospective studies support an high risk prognosis of FISH positive individuals with non-dysplastic histology, brush cytology may be a promising additional diagnostic tool guiding surveillance patterns for BE.