HER2 and TOP2A amplification in a hospital-based cohort of breast cancer patients: associations with patient and tumor characteristics.

Gene amplification is an important factor for altered gene expression in breast cancers. TOP2A-amplification often occurs concomitantly with HER2 amplification, and it has been suggested to be predictive for the response to anthracycline chemotherapy. This study assessed the correlation between HER2 status and TOP2A co-amplification, the possible association of TOP2A single-nucleotide polymorphisms with the frequency of this co-amplification as well as confirmation of association with outcome. HER2 and TOP2A amplification were analyzed in a tissue microarray from a clinical cohort study. Additionally, a common genetic variant (rs13695) in the TOP2A gene was genotyped in germline DNA. HER2 gene amplification was compared with HER2-IHC findings assessed during clinical routine work, and the association between all the biomarkers analyzed and the clinical outcome was determined. As an exploratory aim, rs13695 genotypes were compared with TOP2A amplification status. HER2 amplification was seen in 101 of 628 (16.1 %) and TOP2A amplification in 32 (5.1 %) cancers. No TOP2A amplification occurred without HER2 co-amplification. HER2 amplification was found in 8, 13.6, and 55.1 % of patients with HER2-IHC 0/1+, 2+, and 3+ tumors, respectively. HER2-IHC was not associated with an effect on the prognosis, but HER2-FISH was. There was an association between the rs13695 genotype and TOP2A amplification status (P = 0.03). Although there was a significant correlation between HER2 status determined by IHC and HER2 by FISH, only HER2 gene amplification status by FISH was correlated with outcome indicating greater utility for FISH in routine clinical settings.

HER-2 gene amplification, HER-2 and epidermal growth factor receptor mRNA and protein expression, and lapatinib efficacy in women with metastatic breast cancer.

PURPOSE: Biomarkers from two randomized phase III trials were analyzed to optimize selection of patients for lapatinib therapy. EXPERIMENTAL DESIGN: In available breast cancer tissue from EGF30001 (paclitaxel +/- lapatinib in HER-2-negative/unknown metastatic breast cancer, n = 579) and EGF100151 (capecitabine +/- lapatinib in HER-2-positive metastatic breast cancer, n = 399), HER-2 gene amplification by fluorescence in situ hybridization (FISH), HER-2 mRNA by reverse transcription-PCR (RT-PCR), HER-2 protein expression by HercepTest immunohistochemistry (IHC), epidermal growth factor receptor (EGFR) mRNA level by RT-PCR, and EGFR protein by IHC were analyzed and compared with clinical outcome. HER-2 was determined by FISH in an academic reference/research laboratory and in a large, high-volume commercial reference laboratory. RESULTS: The HER-2 gene was amplified in 47% (344 of 733) and IHC was 3+ in 35% (279 of 798), with significant correlation (P < 0.01) between FISH and IHC. Positive EGFR immunostaining (IHC 1+, 2+, or 3+) in 28% (213 of 761) correlated with EGFR mRNA levels by RT-PCR (r = 0.59; P < 0.01). HER-2 gene amplification/overexpression was associated with improved clinical outcomes (progression-free survival; P < 0.001) in both trials. A significant improvement in outcome was seen in FISH-positive and IHC 0, 1+, or 2+ patients. HER-2 mRNA expression correlated with HER-2 FISH (r = 0.83) and IHC status (r = 0.72; n = 138). No correlation was found between EGFR expression (IHC or mRNA) and responsiveness to lapatinib regardless of HER-2 status. Although a significant correlation with lapatinib responsiveness was observed among "HER-2-negative" breast cancer patients in the large, high-volume commercial reference laboratory, this was not confirmed in the academic reference/research laboratory. CONCLUSIONS: Women with HER-2-positive metastatic breast cancer benefit from lapatinib, whereas women with HER-2-negative metastatic breast cancer derive no incremental benefit from lapatinib.

Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials.

PURPOSE: To critically assess the accuracy and reproducibility of human epidermal growth factor receptor type 2 (HER-2) testing in outside/local community-based hospitals versus two centralized reference laboratories and its effect on selection of women for trastuzumab (Herceptin)-based clinical trials. EXPERIMENTAL DESIGN: Breast cancer specimens from 2,600 women were prospectively evaluated by fluorescence in situ hybridization (FISH) for entry into Breast Cancer International Research Group (BCIRG) clinical trials for HER-2-directed therapies. RESULTS: HER-2 gene amplification by FISH was observed in 657 of the 2,502 (26%) breast cancers successfully analyzed. Among 2,243 breast cancers with central laboratory immunohistochemistry (10H8-IHC) analysis, 504 (22.54%) showed overexpression (2+ or 3+). Outside/local laboratories assessed HER-2 status by immunohistochemistry in 1,536 of these cases and by FISH in 131 cases. Overall, the HER-2 alteration status determined by outside/local immunohistochemistry showed a 79% agreement rate [kappa statistic, 0.56; 95% confidence interval (95% CI), 0.52-0.60], with FISH done by the central laboratories. The agreement rate comparing BCIRG central laboratory 10H8-IHC and outside/local laboratory immunohistochemistry was 77.5% (kappa statistic, 0.51; 95% CI, 0.46-0.55). Finally, HER-2 status, determined by unspecified FISH assay methods at outside/local laboratories, showed a 92% agreement rate (kappa statistic, 0.83; 95% CI, 0.73-0.93), with FISH done at the BCIRG central laboratories. CONCLUSIONS: Compared with the HER-2 status determined at centralized BCIRG reference laboratories, these results indicate superiority of FISH to accurately and reproducibly assess tumors for the HER-2 alteration at outside/local laboratories for entry to clinical trials.

Alteration of topoisomerase II-alpha gene in human breast cancer: association with responsiveness to anthracycline-based chemotherapy.

PURPOSE: Approximately 35% of HER2-amplified breast cancers have coamplification of the topoisomerase II-alpha (TOP2A) gene encoding an enzyme that is a major target of anthracyclines. This study was designed to evaluate whether TOP2A gene alterations may predict incremental responsiveness to anthracyclines in some breast cancers. METHODS: A total of 4,943 breast cancers were analyzed for alterations in TOP2A and HER2. Primary tumor tissues from patients with metastatic breast cancer treated in a trial of chemotherapy plus/minus trastuzumab were studied for amplification/deletion of TOP2A and HER2 as a test set followed by evaluation of malignancies from two separate, large trials for changes in these same genes as a validation set. Association between these alterations and clinical outcomes was determined. RESULTS: Test set cases containing HER2 amplification treated with doxorubicin and cyclophosphamide (AC) plus trastuzumab, demonstrated longer progression-free survival compared to those treated with AC alone (P = .0002). However, patients treated with AC alone whose tumors contain HER2/TOP2A coamplification experienced a similar improvement in survival (P = .004). Conversely, for patients treated with paclitaxel, HER2/TOP2A coamplification was not associated with improved outcomes. These observations were confirmed in a larger validation set, where HER2/TOP2A coamplification was again associated with longer survival when only anthracycline-containing chemotherapy was used for treatment compared with outcome in HER2-positive cancers lacking TOP2A coamplification. CONCLUSION: In a study involving nearly 5,000 breast malignancies, both test set and validation set demonstrate that TOP2A coamplification, not HER2 amplification, is the clinically useful predictive marker of an incremental response to anthracycline-based chemotherapy. Absence of HER2/TOP2A coamplification may indicate a more restricted efficacy advantage for breast cancers than previously thought.