Abstract P4-06-12: Simultaneous phenotypic and genotypic heterogeneity at the cellular level approached by HER2 gene-protein assay (GPA) in the invasive ductal carcinoma of the breast: A new approach for cancer pathobiology
Authors: Osamura RY , Kuwayama A , Katoh M 
Published: August 2014

Abstract
Introduction:
Trastuzumab therapy has been given to the HER2 positive breast cancer (BC) patients (IHC 3+ and/or amplified HER2 gene). Heterogeneity of HER2 expression has been a problem when the recurrence is considered. Recently, the combined HER2 IHC and dual color ISH (DISH) method has been introduced as a gene-protein assay (GPA) (Roche Diagnostics Inc.). This method enables us to analyze heterogeneous expression of HER2 protein and HER2 gene at the individual cell level on the same tissue sections. This study is aimed to elucidate the relevance of GPA in studying the gene-protein(GP) heterogeneity of HER2 status at the individual cells of BCs and further to pursue the cellular GP heterogeneity as a possible cause of stage IV post-therapeutic recurrence.
Materials and Methods:
Formalin-fixed, paraffin-embedded sections from 17 HER2 IHC 3+ cases (5 immediate post-operative cases and 12 recurrence cases) and 11 HER2 IHC 2+ cases (9 immediate post-operative cases and 2 recurrence cases) were analyzed with HER2 GPA using BenchMark ULTRA (Roche Diagnostics K.K.). All slides were scored for HER2 protein expression (0 to 3+) and the ratio of HER2/CEN17 of 100 cells from the following areas: one hot spot and two non-hot spots. HER2 protein and gene status of individual cells were grouped from A to F according to the following definition:
Group A: HER2 IHC positive (3+), DISH positive (HER2/CEN17 ratio ≥2.0)
Group B: HER2 IHC positive (3+), DISH negative (&lt;2.0)
Group C: HER2 IHC equivocal (2+), DISH positive (≥2.0)
Group D: HER2 IHC equivocal (2+), DISH negative (&lt;2.0)
Group E: HER2 IHC negative (0 or 1+), DISH positive (≥2.0)
Group F: HER2 IHC negative (0 or 1+), DISH negative (&lt;2.0)
Results:
HER2 GPA technology clearly demonstrated the HER2 heterogeneity at the individual cell level of invasive BCs. The GP heterogeneity was apparent in the HER2 3+ and 2+ immediate post-operative cases, particularly more obvious with HER2 IHC 2+ cases. For IHC 3+ cases (table 1), groups A and C are prominent with the HER2 GP heterogeneity compared with the other groups. For IHC 2+ cases (table 2), the HER2 GP heterogeneity was more obvious with prominent groups D and F than others.
Table 1: Distribution of groups of HER2 status by GPA Cases with HER2 IHC 3+CasesABCDEFPost-operative79.22.88.407.02.6Recurrent50.83.017.06.17.68.9Post-operative and recurrent cases showing hetelogeneity as individual cell level
For the recurrent cases, the GP heterogeneity was similar but more pronounced comparing to immediate post-operative tumors in IHC 3+ cases, and GP heterogeneity in recurrent cases was more pronounced in IHC 2+ cases.
Conclusions:
HER2 GPA system is an appropriate and essential technology to study the GP heterogeneity at the individual tumor cell level of BCs. We hypothesize that the GPA system may possibly predict the recurrence of post-therapeutic stage IV tumors by the presence of more pronounced HER2 GP heterogeneity.
Table 2: Distribution of groups of HER2 status by GPA Cases with HER2 IHC 2+CasesABCDEFPost-operative01.714.944.78.630.2Recurrent007.00.548.044.5
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-06-12.