Comparison of liquid based cytology and histology for the evaluation of HER-2 status using immunostaining and CISH in breast carcinoma

Abstract

Background: HER-2 amplification is an important prognostic biomarker and treatment determinant in breast carcinoma.

Aims: To correlate immunocytochemical (ICC) expression of HER-2 and gene amplification determined by chromogenic in situ hybridisation (CISH) using liquid based cytology (LBC) with immunohistochemistry (IHC) and CISH using histological samples of the same breast carcinomas.

Methods: Frozen sections and cytobrushings of 103 breast carcinomas were analysed. Four techniques were performed on each tumour: two on LBC samples (ICC, and CISH, both graded as positive, indeterminate, or negative) and two on histological samples (IHC and CISH). Two cell lines (MCF-7, negative; BT 474, positive) were used as controls for cytological analysis. A complementary fluorescence in situ hybridisation technique was carried out in histological samples with low amplification (4-10 dots/nucleus).

Results: Interobserver agreement for the four techniques calculated by the kappa coefficient indicated a substantial agreement. Nine cases failed in cytology because of poor cellularity. Among 94 cases, 19 were amplified; 73, 12, and 9 tumours were scored 0 or 1+, 2+, and 3+, respectively by IHC and 75, 13, and 6, respectively, by ICC. CISH found no amplification in 72 tumours. Correlations between the IHC and CISH results in the histological and cytological samples were always significant.

Conclusions: Her-2 status could be determined in LBC samples and correlated well with reference histological methods using in situ hybridisation. ICC was less reliable because of the presence of the cytoplasmic membrane. However, these results should be confirmed by a large multicentre study.

Figure 1

Immunohistochemistry in invasive ductal carcinoma. HER-2 is characterised by membranous staining. Sections were couterstained with haematoxylin. (A, B) No overexpression of HER-2 (0 and 1+, respectively); (C) intermediate positivity (2+); (D) overexpression (3+).

Figure 2

Examples of chromogenic in situ hybridisation for the HER-2 oncogene and fluorescence in situ hybridisation (FISH) for the chromosome 17 centromere in breast cancer. (A) A tumour with one or two clearly identifiable copies of the HER-2/neu gene (no amplification). (B) This tumour contained six to 10 copies of HER-2/neu gene/nucleus in more than 50% of the cells. (C) Typical high amplification of the HER-2/neu gene appears as a peroxidase positive cluster of gene copies. In these tumours, the FISH technique for the chromosome 17 centromere produced two types of results: (D) two spots showing low amplification, and (E) six to 10 copies/nucleus corresponding to aneuploidy and no amplification.

Figure 3

Immunocytochemistry for the HER-2 protein in liquid based cytology samples from patients with breast cancer. (A) Negative control (MCF7), (B) positive control (BT474), (C) sample with no expression (0), (D) sample with no expression (+1), (E) sample with indeterminate expression (2+), and (F) sample with overexpression of the protein.

Figure 4

CISH analysis of HER-2 gene amplification in thin layer cytology breast cancer samples. (A) Negative control with two spots/nucleus (MCF-7), (B) positive control with more than 10 spots/nucleus (BT474), (C) sample with no amplification (two spots), (D) sample with low amplification (6–10 spots/nucleus), (E) sample with high amplification (> 10 spots/nucleus).