Immunohistochemistry (IHC) and In-Situ Hybridization (ISH) for HER2 on a Single Slide Is Feasible and Allows Direct Comparison at the Individual Cell Level
ES Reisenbichler, A Andea, D Horton, O Hameed. University of Alabama, Birmingham, AL
Background: Although either IHC or ISH may be originally used for HER2 testing in breast carcinoma (BC), repeat testing with a second method is often required for equivocal cases and validation. Given that chromogenic ISH (CISH) is similar to IHC in that conventional bright-field microscopy is used for its interpretation, we investigated the possibility of performing both HER2 IHC and CISH on a single section.
Design: Different sequential and combined protocols for the performance of HER2 CISH (Biocare) and IHC (Dako) on a single slide were evaluated on a BC tissue microarray (TMA) of 10 cases, a HER2-positive control case, and 9 archival BC cases, 3 of which were amplified.
Results: a) CISH after IHC: Multiple evaluations of the HER2-positive control case did not change the 3+ signal obtained when IHC was applied alone; however no CISH signals could be detected. b) IHC after CISH: This produced 100% IHC/CISH concordance in the TMA with three 3+/amplified cases and seven 0 or 1+/non-amplified cases. Evaluation of the HER2-positive control case with 45 min antibody incubation (vs. 30 min for IHC alone) produced a 3+/amplified profile identical to that seen in the individual IHC and CISH tests. c) Combined protocol (antigen retrieval; protease digestion; hybridization; antibody application; dual detection). Applying this on the HER2-positive control case produced the expected 3+/amplified profile (fig); however, there was a slight reduction in the HER2 staining intensity. This was more evident in archival cases where 2 of the 3 amplified cases showed a reduction from the 3+ signal seen by IHC alone to a 2+ signal; the remaining 9 cases had results identical to those in individual IHC and CISH tests.
Conclusions: Although performing CISH after IHC (the usual sequence) did not produce DNA signals, performing IHC after CISH or using a combined protocol to evaluate both on a single section is feasible and permits simultaneous “cell-by-cell” evaluation of HER2 expression and gene amplification status. The main issue is decreased HER2 expression, presumably due to membrane digestion; however, longer antibody incubation may correct this. Additional testing is being performed.
Tuesday, March 23, 2010 1:00 PM
Poster Session IV # 11, Tuesday Afternoon