Significance of HER2 Low-Level Copy Gain in Barrett's Cancer: Implications for Fluorescence In situ Hybridization Testing in Tissues

Abstract

Purpose: HER2 may be a relevant biomarker in Barrett's cancer. We compared three HER2 laboratory methods, standard fluorescence in situ hybridization (FISH), image-based threedimensional FISH in thick (16 Am) sections, and immunohistochemistry, to predict patient outcome. Experimental Design: Tissue microarray sections from 124 Barrett's cancer patients were analyzed by standard FISH on thin (4 Am) sections and by image-based three-dimensional FISH on thick (16 Am) sections for HER2 and chromosome-17, as well for p185HER2 by immunohistochemistry. Correlations with clinical and follow-up data were examined. Results: Only three-dimensional FISH on thick (16 Am) sections revealed HER2 gene copy gain to be associated with increased disease-specific mortality (relative risk, 2.1; 95% confidence interval, 1.06-4.26; P = 0.033). In contrast, standard FISH on thin (4 Am) sections and immunohistochemistry failed to predict clinical outcome. Low-level gain of HER2 occurred frequently in Barrett's cancer (z2.5-4.0 HER2 copies, 59.7%; HER2-to-chromosome-17 ratio, z1.1-2.0; 61.2%) and defined a subpopulation for patient outcome as unfavorable as HER2 gene amplification [disease-free survival, P = 0.017 (HER2 copies)]. This low-level group was neither definable by standard FISH nor immunohistochemistry. No prognostic significance was found for chromosome-17 aneusomy. Conclusions: Low-level copy gains of HER2 define a biologically distinct subpopulation of Barrett's cancer patients. Importantly, these subtle copy number changes are not reliably detected by standard FISH in thin (4 Am) tissue sections, highlighting a thus far unrecognized weakness in HER2 FISH testing. These results should be taken into account for accurate evaluation of biomarkers by FISH and for HER2 FISH testing in tissue sections.Significant strides continue to be made in the treatment of patients with Barrett's cancer (Barrett's esophagus -associated adenocarcinoma), which is now the most common esophageal cancer and which incidence is increasing faster than that of any other malignancy in the United States (1 -3). At this time, early detection and complete surgical resection are the surest ways to cure esophageal cancer (3). Thus, new therapeutic targets and better prognostic markers for patient stratification are needed.Accurate determination of the status of HER2 in cancer provides significant insight into patient prognosis and may also inform selection of chemotherapeutic treatments. The effect of analyzing HER2 status by either fluorescence in situ hybridization (FISH) or immunohistochemistry has been investigated and also reviewed in extensive meta-analyses, supporting that FISH provides a more powerful prognostic indicator (4 -7). This prompted us to investigate the genomic basis of HER2 expression and its prognostic implication in a cohort of Barrett's cancer patients. However, similar to immunohistochemistry methods, not all FISH methods are alike. To date, most FISH studies for standard HER2 testing of paraffinembedded tissue have been d...

Assessment of ErbB2 (Her2) in oesophageal adenocarcinomas: summary of a revised immunohistochemical evaluation system, bright field double in situ hybridisation and fluorescence in situ hybridisation

“…Nevertheless, the majority of cases were contained in both tissue microarrays so that correlation between immunohistochemistry, bright field double in situ hybridisation and fluorescence in situ hybridisation could be performed in approximately 100 cases. Discrepancies between the various in situ hybridisation methods and immunohistochemistry in our study may not only be caused by methodical or technical differences or the 'grey zone' of low-level gains 12 but also represent the phenomenon of intratumoral heterogeneity of ErbB2 expression in gastro-oesophageal adenocarcinomas, [23][24][25][26] because the cores of the donor blocks were obtained from slightly different tumour areas. Therefore, a heterogeneous distribution of ErbB2-positive cell clones may also be responsible for those divergent findings.…”

“…18,21,22 This phenomenon frequently occurs in oesophagogastric carcinomas 1,3,23 and can readily be recognised by this method. We have compared bright field double in situ hybridisation data with fluorescence in situ hybridisation data from a previously published study 12 and found a significant correlation between bright field double in situ hybridisation and both conventional fluorescence in situ hybridisation and the more complex but more sensitive 3D fluorescence in situ hybridisation. For breast cancer, similar observations were reported by others who described a high concordance in the assessment of ErbB2 amplification between fluorescence in situ hybridisation and the light-microscopy-based techniques of silver in situ hybridisation and bright field double in situ hybridisation, respectively, with advantages in terms of handling, orientation within the slides and in cases of intratumoral heterogeneity for bright field double in situ hybridisation.…”

“…Fluorescence in situ hybridisation analysis was done on the first tissue microarray, which contained cores from 124 tumours 11,12 with three cores per tumour. Results of this study were originally published in 2006 and 2007.…”

“…Additional data from conventional fluorescence in situ hybridisation using 4 mm tissue microarray sections and three-dimensional (3D) fluorescence in situ hybridisation using 16 mm tissue microarray sections were obtained from a previously published study by our group. 12 For the purpose of this study, cases with an ErbB2/Chr17 ratio of Z2 based on either conventional or 3D fluorescence in situ hybridisation were classified as ErbB2 amplified. ErbB2 amplification was further subclassified into low-level amplification (ErbB2/Chr17 ratio 2-3) and high-level amplification (ErbB2/Chr17 ratio 43), consistent with published studies.…”

“…The rate of ErbB2 overexpression/amplification in these cancers has been reported at approximately 20-25%, depending on the method of evaluation and the definition used. [11][12][13][14][15] In terms of the relatively high rate of chemotherapy resistance in oesophageal adenocarcinomas, 16,17 additional therapeutic options, such as ErbB2-targeting are of high demand for patients with advanced or metastatic disease.…”