HER2 status for all samples was determined by conventional FISH and nuclei microarray FISH. Signals for both HER2 and chromosome 17 centromere were clearly detected using the nuclei microarray method (Figure 1
). Separate signals could be counted without difficulty for non-amplified, equivocal, and amplified HER2 samples. Of the 152 cases analyzed, 42 were found to be amplified both by conventional FISH and Nuclei Microarray FISH (NMFISH) (ratio of HER2
/CEP17 more than 2.2), giving an amplification rate of 27.6%.
Of the 152 cases, the NMFISH method detected HER2 amplification (HER2 ratio above 2.2) in 44 cases, equivocal (ratios from 1.8 to 2.2) in 22 cases and 86 cases showed non-amplified (ratios less than 1.8), the conventional FISH method detected HER2 amplification in 42 cases, equivocal in 23 cases and 87 cases showed non-amplified HER2. The agreement between NMFISH and conventional FISH was almost perfect: 95.4% (145/152), κ coefficient = 0.920 (Table 1
There was no significant difference between the two methods using the McNemar–Bowker Test (P = 0.333).
There were two discrepant cases that showed non-amplification by nuclei microarray FISH but equivocal amplification by conventional FISH. Three cases showed equivocal by nuclei microarray FISH but non-amplified by conventional FISH and two showed amplified by nuclei microarray FISH but equivocal amplification by conventional FISH. The HER2
/CEP17 ratios of these seven cases are detailed in Table 2
/CEP17 ratios detected with the two diagnostic methods were very similar and close to the cut-off value. This may have caused the differences in diagnoses.
Trastuzumab therapy has been used for the treatment of metastatic breast cancer for some years. Due to the development of adjuvant trastuzumab therapy, pathological laboratories are currently requested to perform HER2 status assays for all newly diagnosed breast cancers. The ASCO/CAP guidelines recommend the HER2 status to be optionally determined by IHC or FISH. In most laboratories, IHC is done first. In cases where the primary immunohistochemical result is equivocal at the protein level, such as an IHC score of 2+, faint staining or other artifacts, an additional test is strongly recommended to clarify the HER2 status at the genomic level to determine amplification or nonamplification. Thus, the need for reliable diagnostic and cost-effective HER2 gene tests is rapidly increasing.
Tissue microarrays, also called tissue chips, are a novel technology invented by Konenen et al
], based on cDNA microarrays in 1998. It is a high throughput and resource conserving technology where tens of thousands of typical minute cylindrical tissue samples or cells from hundreds of different tumors are transferred to a new paraffin block. Tissue microarrays can be used in the detection of DNA, RNA or protein in various clinical or basic research areas [20
Christopher et al
] introduced methods to make cultured cells into microarrays. This inspired us to make nuclei microarray with nuclei extracted from the paraffin embedded tissues for the FISH detection. The present study proved this method was feasible for the detection of HER2
gene amplification in breast cancer.
During the process of making the nuclei microarray, 30-μm thick paraffin sections were cut for the extraction of nuclei instead of using the 4-μm thick sections used during the conventional FISH detection. This ensured that the nuclei were intact without being sectioned and the genetic DNA material was retained.
In the majority of the cases, the FISH method was performed successfully on nuclei microarray and almost perfect agreement with the FDA-approved HER2 FISH pharmDx method was revealed. Similar reagents and handling in the pretreatment, denaturation, hybridization and stringent washing steps were used in the two methods. One hundred samples can be detected simultaneously using this method with the same amount of reagents previously needed for one entire specimen, greatly reducing the cost.
The nuclei microarray FISH technology is advantageous for its high efficiency and low background compared to the conventional FISH using paraffin embedded tissue sections. The entire tissue section was used for the extraction of nuclei from multiple areas of the tumor. Each microarray disk contains more than five hundred cells [18
], which are sufficient for the FISH detection.
During the conventional FISH detection using paraffin embedded tissue sections, variations can occur due to loss of the genetic DNA material occurring when the tissue blocks were sectioned. In the present study, we did not find this discrepancy since the ratios between HER2 signal and CEP17 were used to evaluate the HER2 gene amplification in breast cancer. The probability of the signal losses for HER2 and CEP17 due to sectioning is the same in this experiment. Thus, the nuclei microarray FISH and conventional FISH are both applicable for detecting HER2 gene amplification in breast cancer.
The present study compared nuclei microarray FISH and conventional FISH in detecting HER2 gene amplification in breast cancer. The McNemar–Bowker test revealed that there was no significant difference between the two methods (P = 0.333). κ test revealed there was almost perfect agreement between the two methods (κ coefficient = 0.920). There are seven cases with discrepant results while the ratios are close to the diagnostic cut-off value. This may be caused by the heterogeneity of the tissue specimens.