Out of the Dark into the Light: Brightfield Dual In Situ Hybridization as a Modality for Assessing HER2 Gene Amplification in Invasive Urothelial Carcinoma
Paolo Cotzia, Alessandro Bombonati, Raymond O'Neill, Daniel Jaller, Rossitza Draganova-Tacheva, Charalambos Solomides, Stephen Peiper, Peter McCue, Ruth Birbe. Thomas Jefferson University Hospital, Philadelphia, PA
Background: HER2 gene amplification has been reported as a survival predictor in muscle-invasive urothelial carcinoma (UCa). The current modality for assessing the HER2 positivity is the one used in breast cancer. It follows a two step process with HER2 immunohistochemical stain (IHC) and confirmatory fluorescent in situ hybridization (FISH). Tumor heterogeneity and polysomy 17, commonly found on UCa, limits the usefulness of these techniques. FISH requires a fluorescence microscope and the signal attenuates with time. More than “working in the dark”, FISH forces the pathologist to work in the dark on a histologic level as well. Brightfield HER2 dual color in situ hybridization (HER2 Dual ISH) ameliorates these limitations, is permanent, storable, and less technically demanding. It enables the diagnosis of HER2 amplification and polysomy 17 in the context of morphology.
Design: After IRB approval, we searched retrospectively invasive UCa (resection and cystectomy) and benign controls. Seventy invasive high grade UCa and 5 benign bladders were selected. The diagnosis was confirmed with the H&E slides. A 2 mm tissue core microarray was performed from the formalin fixed-paraffin embedded tissue blocks using the Veridiam tissue arrayer. HER2/neu IHC and HER2 Dual ISH tests were performed using the anti-HER2/neu (4B5) rabbit monoclonal primary antibody and the INFORM HER2 Dual ISH DNA Probe Cocktail according to the manufacturer's protocols (Ventana Medical Systems). The results were analyzed by three pathologists following the breast cancer guidelines. A chromosome (Chr) 17 average above 2.2 signals per 20 cells was diagnosed as polysomy 17 on the HER2 Dual ISH test.
Results: HER2 gene was amplified in 4 of 70 (5.7%) invasive UCa but not on the 5 benign bladders (0%). HER2 was overexpressed (3+) in 18 UCa cases (25.7%); including all 4 amplified cases. 77.7% of the HER2 3+ cases were not amplified. 26 UCa cases had HER2 2+ indeterminate results (37%), none of them were amplified; however 20 of 26 (76%) had polysomy 17. Polysomy 17 was present on 55 of the 70 UCa cases (78%) including 3 of the HER2 Dual ISH amplified cases.
Conclusions: HER2 overexpression and Dual ISH amplification have 100% concordance in UCa. Commonly HER2 overexpression is due to polysomy 17 rather than HER2 gene amplification limiting the value of HER2 IHC testing. HER2 Dual ISH might be a superior single test to detect HER2 gene amplification than the classic two-step process IHC and FISH in invasive UCa.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 263, Wednesday Morning