High resolution representational oligonucleotide microarray analysis (ROMA) suggests that TOPO2 and HER2 co-amplification is uncommon in human breast cancer

Background Clinical studies relating TOPO2A expression or amplification by FISH have yielded inconsistent results regarding prediction of benefit from various drug therapies, particularly in the setting of HER2 amplification by FISH. Because HER2 and TOPO2A are relatively small genes that are close to each other, and because the relevant commercially-available FISH probes hybridize to DNA sequences beyond the genes of interest, there is significant potential for false positive results. We therefore conducted an exploratory study comparing HER2 and TOPO2A amplification by ROMA and FISH. Methods: Forty-two archived formalin-fixed paraffin-embedded primary breast cancer specimens were pre-selected to contain 36 HER2 amplified and 6 HER2 non-amplified cases by FISH. These specimens were evaluated for HER2 and TOPO2A amplification by FISH at Memorial Sloan-Kettering Cancer Center and by ROMA at Cold Spring Harbor Laboratory in a double-blinded experiment. Two HER2 amplified by FISH specimens proved inevaluable for technical reasons. The results for the remaining 40 evaluable specimens were then compared. Results: Of the 40 evaluable specimens, the 6 cases selected as HER2 non-amplified by FISH were HER2 non-amplified by ROMA and TOPO2A non-amplified by both FISH and ROMA. Thirty-two specimens were HER2 amplified by both FISH and ROMA. Two specimens with HER2 FISH results of 2.2 and 2.4 did not demonstrate focal amplification by ROMA. Twenty-five (74%) of the 34 specimens with HER2 amplification by FISH were TOPO2A non-amplified by both FISH and ROMA (Table 1). However, only 2 (22%) of the 9 specimens with HER2 and TOPO2A co-amplification by FISH demonstrated TOPO2A amplification by ROMA. Conclusions: Our results suggest that by ROMA, the determination of HER2 status (amplified and non-amplified) and TOPO2A non-amplification by FISH is accurate. The absence of TOPO2A amplification in HER2 non-amplified breast cancer is consistent with published reports. We also confirmed the published frequency of HER2 and TOPO2A co-amplification by FISH. However, we found that co-amplification of HER2 and TOPO2A by ROMA is uncommon, which suggests that commercially-available FISH probes over-estimate the frequency of TOPO2A amplification in HER2 amplified cases. Studies evaluating the clinical implications of these findings are underway