Analysis of HER2 by chromogenic in situ hybridization and immunohistochemistry in lymph node–negative breast carcinoma: prognostic relevance

Summary

In patients with lymph node–negative breast carcinoma (LNNBC), the prevalence of HER2 overexpression and gene amplification and their prognostic value have not been extensively evaluated. We examined 162 patients with LNNBC with complete follow-up. Immunohistochemistry (IHC) for HER2, Ki67, and p53 was performed. HER2 gene status was analyzed by chromogenic in situ hybridization (CISH) and discordant cases by fluorescence in situ hybridization. HER2 overexpression was seen in 24.7% of cases (40/162) and amplification by CISH in 17.6% (28/159). Agreement between IHC and CISH was achieved in 147 (92.5%) cases. Amplification was seen in 21 (100%) of 21 (3+), 6 (35.3%) of 17 (2+), and 1 (0.6%) of 121 (0-1+) tumors. Fluorescence in situ hybridization detected 3 (1.8%) additional cases. HER2 overexpression and amplification were present in tumors of high grade, with necrosis and lymph-vascular invasion (LVI) (all P < .027). In addition, amplified tumors showed Ki67 of more than 20% and p53 overexpression (P < .05). By univariate analysis, shorter disease-free survival (DFS) and overall survival (OS) were seen for patients with tumors showing HER2 amplification, LVI, and Ki67 of more than 20% (P < .05) (Kaplan-Meier). However, the multivariate analysis (Cox regression) demonstrated only Ki67 as an independent prognostic factor for both DFS (P = .017) and OS (P = .010), and as a trend for HER2 gene status (OS, P = .087) and LVI (DFS, P = .11; OS, P = .063). We conclude that IHC is a reliable method for detecting HER2 expression that can be complemented by CISH in nondefinitive cases (2+). Moreover, CISH is a valuable tool for the assessment of HER2 gene status with potential prognostic value and, therefore, in clinical decision making for treatment of high-risk LNNBC.

Introduction

Patients with lymph node–negative breast carcinoma (LNNBC) are considered to have a good prognosis. Nevertheless, about one third will develop metastases. Although useful prognostic information can be obtained from clinicopathologic data, the identification of molecular genetic alterations for better selection of patients who will need additional or specific treatment has become relevant. Among them, HER2 has been proved to have a role in the pathogenesis of breast carcinoma and in a significant number of human tumors [1], [2]. HER2 gene (17q21) encodes a 185-kd transmembrane glycoprotein with tyrosine kinase activity that functions as a growth factor receptor. In normal cells, protein expression is primarily regulated by transcription activation and gene amplification. The oncogenic activation results in abnormally large amounts of the nonmutated receptor on the cell surface, which in turn may lead to autoactivation of the tyrosine kinase domain, activation of signal transduction pathways, and cellular transformation or proliferation [3]. In breast carcinoma, HER2 overexpression and/or amplification is found between 20% and 30% of the tumors [1], [4], [5], [6], [7] and can occur early in the development of the disease [8].

Recently, analysis of HER2 status has become a common practice in surgical pathology laboratories. Several methods have been proved to be useful for the assessment [9], but most of them are beyond the scope of the majority of laboratories due to technical and economic reasons [4], [10], [11]. Furthermore, consensus regarding the best method, reagents, or cut-off points to define HER2 status has not yet been reached. In clinical practice, the most commonly used methods are immunohistochemistry (IHC) for the protein expression and fluorescence in situ hybridization (FISH) for the evaluation of gene amplification. On one hand, IHC assay reliability has been questioned because of the high rate of discordance among the studies [5], [6], [7], [11]. On the other hand, FISH analysis is seen as the gold standard method for detecting HER2 amplification, with 98% sensitivity and 100% specificity when compared with other assays [10], [12]. However, it is not practical for routine histopathologic laboratories because of the additional expensive equipment and its time-consuming technique [4]. Recent studies indicate that the level of amplification provides more meaningful prognostic information than IHC [12].

Currently, there is an increasing demand for new methods to accurately assess HER2 gene status which can be performed in routine clinical practice. Chromogenic in situ hybridization is a relatively new technique that enables the detection of HER2 gene copies with a conventional peroxidase reaction [13]. It appears to be an attractive method for clinical HER2 analysis owing to its specific targeting of neoplastic cells and retrospective potential. Comparison of chromogenic in situ hybridization (CISH) results with IHC or FISH has shown good correlation in recent studies [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]. Nevertheless, little data are available regarding the clinical value of CISH [2], [18], [24], [25], [26].

In LNNBC, the prognostic relevance of HER2 has been controversial. Although some investigators have found that overexpression and/or amplification confers a worse prognosis, others have been unable to confirm these results [5], [18], [24], [25], [26], [27], [28]. In addition, the value of Ki67 and p53 expression, common markers of cellular proliferation and apoptosis, in predicting outcome in these early-stage patients has not been extensively studied [25], [28], [29]. Therefore, we evaluated in a series of patients with LNNBC the prevalence and prognostic relevance of HER2 expression by IHC and gene amplification by CISH, as well as the correlation with Ki67 and p53 expression. We found that both HER2 overexpression and amplification were associated with adverse prognostic factors, but only the level of amplification correlated with the outcome, whereas Ki67 had an independent prognostic value. We concluded that IHC can be used as the first-line screening assay in routine assessment of HER2, followed by CISH to confirm indeterminate cases. Fluorescence in situ hybridization should be performed in cases unresolved by the 2 previous methods. Chromogenic in situ hybridization is a promising technique that can be implemented in routine clinical practice and is useful for selecting prognostic groups of patients.