HER2 FISH testing

In the assessment of HER2 status, immunohistochemistry (IHC) was applied and followed by FISH in selected cases, ie, if the results of IHC were equivocal (the expression assessed as 2+) (Figure 1A). In the investigated group, we additionally assessed status of HER2 gene in cases with 3+ IHC score, which constituted most of the patients (77.4%) (Figure 1B). HER2 gene copy number was investigated by FISH using PathVysion HER-2 DNA Probe Kit II (Vysis, Abbott Laboratories) according to manufacturer’s protocol. The PathVysion HER-2 DNA Probe Kit consists of two labeled DNA probes. The LSI HER-2/neu probe that spans the HER2 gene (17q11.2-12) is labeled in SpectrumOrange, while the CEP17 probe that is labeled in SpectrumGreen hybridizes to the alpha satellite DNA located at the centrom-ere of chromosome 17 (17p11.1–q11.1). Inclusion of the CEP17 probe allows detection of the relative copy number of HER2 gene.

Visualization of HER2 amplification (FISH) and HER2 protein expression (IHC). (A) HER2 expression score 2+, (B) HER2 expression score 3+, (C) example of low level of HER2 gene amplification, square outlined by dotted line is magnified in left bottom corner, (D) example of high level of HER2 gene amplification, (E) example of HER2 amplification with polysomy, and (F) HER3 expression. (A, B, and F) 20× magnification, (C, D, and E) 100× magnification.

Abbreviations: FISH, fluorescence in situ hybridization; HER2, human epidermal growth factor receptor type 2; IHC, immunohistochemistry.

FISH signals were assessed with an Olympus fluorescence microscope BX41 by two independent investigators. Using 100× objective lens, 20–40 interphase tumor nuclei were examined for each specimen, and the numbers of fluorescent signals within tumor cells from HER2 gene and chromosome 17 centromere were recorded.

The slides were evaluated according to ASCO/CAP 2013 recommendations.3 The status of HER2 gene was defined as follows: 1) amplification of HER2 gene was defined as HER2/CEP17 ratio ≥2 (Figure 1C and D) or HER2 gene copy number ≥6 signals per cell and 2) polysomy was defined as average number of signals from centromere of chromosome 17 per cell ≥3 (Figure 1E).