Overexpression of the HER2 protein in breast cancer is most often the result of HER2 gene amplification on the q arm of chromosome 17. Standard testing methods include analysis of HER2 protein expression on the cell membrane by IHC and/or evaluation of HER2 gene copy number by in situ hybridiztion (ISH), most commonly fluorescence (FISH), but also silver (SISH) or chromogenic (CISH), using DNA-based probes targeting the HER2 gene locus and chromosome 17 centromere (CEP) . HER2 protein overexpression and gene amplification are prognostic markers for aggressive tumors and predictive of response to the drugs trastuzumab (Herceptin®) and lapatinib (Tykerb®). Accurate and definitive reporting of HER2 status is thus essential for appropriate treatment planning in newly diagnosed cases. Yet despite the clinical need for accurate determination of HER2 status, it is estimated that approximately 20% of current HER2 testing results may be inaccurate. This inaccuracy has been most often attributed to multiple preanalytic, analytic, and postanalytic variables inherent to the mechanics of performing the test in a clinical laboratory .
In addition to testing inaccuracies, clinicians are also faced with treatment dilemmas resulting from cases that are reported as "equivocal" after testing by IHC and FISH have been completed. These cases are either 2+ by IHC and/or have a HER2/CEP17 ratio between 1.8 and 2.2. However, there are also instances where the results of FISH and IHC are discordant such that one test is reported as positive (amplified) and the other as negative (unamplified). Although the numbers of these equivocal and discordant cases vary widely between laboratories, it is estimated in some studies to be as high as 20% of cases .
The prevalence of inaccurate, discordant, and equivocal HER2 results has lead to a reexamination of the adequacy of existing methods to accurately detect copy number changes involving the HER2 gene, particularly in the setting of complex chromosome 17 rearrangements. Recent genome wide array CGH studies have revealed that complete polysomy 17, which had previously been reported as prevalent in breast cancer, is actually a rare event [4, 5]. These and similar extended FISH studies of chromosome 17 in breast cancer have additionally shown that amplifications of the pericentromeric region are common occurrences in both HER2 positive and HER2 negative cases [6, 7]. The complexity of these chromosome 17 pericentromeric rearrangements detected by both array CGH and FISH analysis has brought into question the accuracy of reported HER2/cep 17 ratios in cases where complex segmental aneusomy of chromosome 17 is present. This observation has lead to the hypothesis that unsuspected chromosome 17 copy number changes may be contributing to the high percentage of inaccurate and equivocal results for HER2 status in breast cancer.
The recent introduction of array-based molecular karyotyping into some clinical laboratories provides an alternative method for clinical genomic evaluation of oncology samples [8, 9]. Array-based chromosomal analysis combines the precision of locus-specific FISH with a complete, whole-genome view of the chromosome complement of a cell, giving clinicians both an accurate assessment of copy number changes involving specific genes, as well as an evaluation of the relevant chromosomes in the context of the entire genome. There is growing evidence that copy number evaluation of both the HER2 gene and chromosome 17 are of prognostic significance in breast cancer, and that aneusomy 17 with or without HER2 gene amplification is associated with poor prognostic factors [10–12].
The aim of this study was to resolve the HER2 gene and chromosome 17 status in cases of invasive ductal carcinoma, where gene copy number and chromosome status were equivocal and/or discordant (based on previously performed IHC and FISH analysis), by using a clinical array CGH assay for copy number evaluation of multiple loci along chromosome 17.