Combined detection of Her2/neu gene amplification and protein overexpression in effusions from patients with breast and ovarian cancer

Abstract

Purpose: Her2/neu protein overexpression and gene amplification is found in 20-30% of breast cancer patients and correlates with poor clinical outcome. Patients who profit from anti-Her2/neu- therapy are routinely selected by examination of tumour specimens using immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) separately. Many studies found a good correlation between both methods for score 1+ samples and for score 3+ samples, but not for score 2+ samples. In this study, we examined pleural and ascitic effusions with a combined approach using IHC and FISH on the same cells, under the following aspects: (1) frequency of Her2/neu protein expression and gene amplification in effusions; (2) correlation between score of protein expression and gene amplification; (3) impact of chromosome 17 polyploidy on Her2/neu protein expression.

Methods: We examined 31 effusions from patients with breast cancer and 4 effusions from patients with ovarian cancer. Cytospins were analysed by IHC using two anti-Her2/neu antibodies and subsequently analysed by FISH with Her2/neu/CEP17 probes. Amplification was defined as: (1) Her2/neu gene copy number of more than 4 and (2) Her2/neu/CEP17 ratio of 2.0 or greater.

Results: A system combining IHC and FISH was developed and 35 effusion specimens were examined. As much as 25 of them were scored as positive. All of them contained cells with heterogeneous scores. A total of 18 of the samples contained cells with scores that ranged from 0 to 3+. In the other samples scores ranged from 0 to 1+ or 0 to 2+. Cells were analysed for Her2/neu gene amplification and chromosome 17 ploidy with regard to their scores. As much as 15 of all samples had mean Her2/neu copy numbers of >4, but only 12% (n = 3) of the positive samples were amplified according to Her2/neu/CEP17 ratio. Only 22, 9% (n = 8) were polyploid (mean CEP17 > 4); but 65, 7% (n = 23) of all specimens contained single polyploid cells. In some cases up to 100% of the score 3+ cells showed chromosome 17 polyploidy. Here protein overexpression might be caused by polyploidy rather than by gene amplification. In some samples, we found single cells with gene amplification but without protein expression and cells without amplification but with protein overexpression.

Conclusion: The combination of IHC and FISH allows a differentiated analysis of single cells, which is especially important for effusions that are composed of heterogeneous cells. Therefore, cells with high gene amplification and/or protein overexpression can be detected and analysed even if their amount in the sample is small. Chromosome 17 polyploidy is important in some cases but this should be further examined on a larger series.

Fig. 1

Simultaneous detection of Her2/neu genes and proteins using immunohistochemistry and FISH on the same cells. Cytospins with HTB20 and K562 (controls) were prestained with antibody TAB 250 and submitted to FISH with Her2/neu probe (Spectrum Orange labelled) and CEP17 probe (Spectrum Green labelled) (magnification 1,000×). a Light microscopic image. b Fluorescence microscopic image of the same cells. c Fluorescence microscopic image in combination with light microscopy. d Fluorescence microscopic image (Spectrum Orange labelled)

Fig. 2

Simultaneous detection of Her2/neu genes and proteins using immunohistochemistry and FISH on the same cells. Cytospins with Pleu 265a were prestained with antibody TAB 250 and submitted to FISH with Her2/neu probe (Spectrum Orange labelled) and CEP17 probe (Spectrum Green labelled) (magnification 1,000×). a–c Light microscopic image (score 3+). Fluorescence microscopic image (Her2/neu, orange). Fluorescence microscopy (chromosome 17, green; Her2/neu, orange). d–f Light microscopic image (score 0). Fluorescence microscopy (chromosome 17, green). Fluorescence microscopy (Her2/neu, spectrum orange)

Fig. 3

Percentage of amplified and polyploid cells in different score-groups of three selected effusion samples. a Pleu 539, b Asc 384, c Pleu 491