Topoisomerase 2A gene amplification in breast cancer. Critical evaluation of different FISH probes

Abstract

The HER2 amplicon on chromosome 17q is variable in size and occasionally includes Topoisomerase 2A (TOP2A) at 17q21-22. It has been suggested that TOP2 coamplification, not HER2 amplification on chromosome 17q11.2-12, is a useful predictive marker of response to anthracycline-based chemotherapy in breast cancer patients. Given the significant toxicities of anthracyclines, the detection methods of TOP2A gene amplifications have to be standardized. We determined TOP2A gene alterations using two different fluorescence in situ hybridization (FISH) DNA probes. HER2 amplifications were identified with the PathVysion TM probe. TOP2A status of 42 HER2 amplified breast cancers was tested by FISH with PathVysion TM covering 160 kb and DAKO pharm DX TM covering 228 kb of the TOP2A amplicon. TOP2A protein expression was tested by immunohistochemistry. Multiplex-ligation dependent probe amplification (MLPA) was performed retrospectively in cases showing discrepancies. TOP2A was amplified in 15 of 42 cases (35%) with DAKO pharm DX TM and in 11 of 42 cases (26%) with PathVysion TM . In all four discrepant cases, MLPA showed no TOP2A amplification, but instead amplification of an upstream region including HER2. TOP2A was deleted in the same seven of 42 carcinomas (17%) with both probes. TOP2A protein expression was detected in all 42 tumours (100%) with high intratumoral heterogeneity. TOP2A amplification rate depends on the length of the hybridized probes for the TOP2A locus. Because TOP2A, not HER2, is a target of anthracyclines, non-overlapping DNA probes should be used to evaluate any associations between such alterations and response to anthracycline-based chemotherapy.

TOP2Agene copy number change in breast cancer

“…Furthermore, a short DNA probe for TOP2A was used to avoid overlap with HER2. 21 This may in part account for the low number of TOP2A -amplified cases in this study compared with previous studies and may reflect the true frequency of this finding.…”

Topoisomerase II-alfa gene as a predictive marker of response to anthracyclines in breast cancer

“…Recently, Varga et al show that using three different commercial kits to detect TOP2A amplification by in situ hybridization discrepant results were found, suggesting that the difference rate of TOP2A amplification is due to the different length of the DNA probes hybridizing to the TOP2A (17q21-q22). They demonstrated that larger TOP2A DNA probe (as in DAKO pharm DX TM with 228 kb) most likely overlaps with the HER2 gene region resulting in overdetection [26]. This is not the case in our series where we used a smaller probe with 67 kb, which could also explain the lower incidence of co-amplification of HER-2.…”

“…However we have only 10 cases with HER2 amplification, and 5 of those showed TOP2A alterations. In fact the amplification rate of the TOP2A gene in HER2 amplified breast carcinoma has been reportedly with variations from 7 to 50% [26]. Regarding the biological characteristics of the tumors, it was found that TOP2A altered tumors were more aggressive in terms of nuclear grade, more frequently with HER2 amplification and inflammatory type.…”

Topoisomerase 2α status in invasive breast carcinoma – comparison of its clinical value according to immunohistochemical and fluorescence in situ hybridization methods of evaluation

“…Outcomes of results both at the level of evaluation of TOP2A as well as at the level of interpretation are extremely different [24][25][26][27][28][29][30][31][32][33]. If biology of breast carcinoma is similar around the world, then methods of TOP2A evaluation, criteria of interpretation and selection of patients are responsible for the differences [34][35][36][37][38][39][40][41]. We put emphasis on criteria of interpretation, and decided to create out own system of We found in our study that cases with positive TOP2A status measured by the IHC method for TOP2A protein presented a statistically significantly better response to therapy which included anthracyclines as one of the chemotherapeutics (AC program in our study) in neoadjuvant therapy measured by relapses (DFS).…”