Clinical array-based karyotyping of breast cancer with equivocal HER2 status resolves gene copy number and reveals chromosome 17 complexity

Abstract

Background: HER2 gene copy status, and concomitant administration of trastuzumab (Herceptin), remains one of the best examples of targeted cancer therapy based on understanding the genomic etiology of disease. However, newly diagnosed breast cancer cases with equivocal HER2 results present a challenge for the oncologist who must make treatment decisions despite the patient's unresolved HER2 status. In some cases both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are reported as equivocal, whereas in other cases IHC results and FISH are discordant for positive versus negative results. The recent validation of array-based, molecular karyotyping for clinical oncology testing provides an alternative method for determination of HER2 gene copy number status in cases remaining unresolved by traditional methods.

Methods: In the current study, DNA extracted from 20 formalin fixed paraffin embedded (FFPE) tissue samples from newly diagnosed cases of invasive ductal carcinoma referred to our laboratory with unresolved HER2 status, were analyzed using a clinically validated genomic array containing 127 probes covering the HER2 amplicon, the pericentromeric regions, and both chromosome 17 arms.

Results: Array-based comparative genomic hybridization (array CGH) analysis of chromosome 17 resolved HER2 gene status in [20/20] (100%) of cases and revealed additional chromosome 17 copy number changes in [18/20] (90%) of cases. Array CGH analysis also revealed two false positives and one false negative by FISH due to "ratio skewing" caused by chromosomal gains and losses in the centromeric region. All cases with complex rearrangements of chromosome 17 showed genome-wide chromosomal instability.

Conclusions: These results illustrate the analytical power of array-based genomic analysis as a clinical laboratory technique for resolution of HER2 status in breast cancer cases with equivocal results. The frequency of complex chromosome 17 abnormalities in these cases suggests that the two probe FISH interphase analysis is inadequate and results interpreted using the HER2/CEP17 ratio should be reported "with caution" when the presence of centromeric amplification or monosomy is suspected by FISH signal gains or losses. The presence of these pericentromeric copy number changes may result in artificial skewing of the HER2/CEP17 ratio towards false negative or false positive results in breast cancer with chromosome 17 complexity. Full genomic analysis should be considered in all cases with complex chromosome 17 aneusomy as these cases are likely to have genome-wide instability, amplifications, and a poor prognosis.

Figure 1

Reference Values. A: Objective reference values for determination of HER2 gene copy number, B: Subjective determination by visualization of chromosome 17 ratio plot.

Figure 2

HER2 positive cases with ratio-skewing by FISH. HER2 positive cases showing co-amplification of the centromere and HER2 gene.

Figure 3

HER2 positive cases with aneusomy and polysomy. A: HER2 positive cases with complex chromosome 17 rearrangements B: Case of HER2 positive polysomy 17 which was positive by IHC and negative by FISH.

Figure 4

HER2 negative cases with aneusomy. HER2 negative cases showing complex pericentromeric rearrangements.

Figure 5

HER2 negative cases with aneusomy and monosomy. A: HER2 negative cases with centromeric loss B: Case showing complete monosomy 17.

Figure 6

Proposed algorithm for establishment of HER2 status in breast cancer samples by protein expression and genomic analysis. IHC studies for HER2 protein expression are performed as part of the diagnostic workup and confirmed or resolved by FISH. If FISH and IHC results are concordant, or equivocal IHC results are resolved by FISH, no further testing is done. Equivocal cases by FISH and cases with discordant IHC/FISH results are resolved by array CGH.