Expression of HER 2 and the Coamplified Genes GRB 7 and MLN 64 in HumanBreastCancer : QuantitativeReal-timeReverseTranscription-PCRas a Diagnostic Alternative to Immunohistochemistry and Fluorescence In situ Hybridization

Purpose: Accurate testing of HER2 is centrally important for breast cancer therapy and prognosis. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are current standard testing methods. As a potential alternative for assessment of HER2 , we explored quantitative real-time reverse transcription-PCR (RT-PCR), a fast and inexpensive method yielding quantitative results insensitive to interobserver variability and amenable to standardized scoring. Experimental Design: We assessed HER2 status at the DNA, mRNA, and protein levels with FISH, quantitative RT-PCR, and IHC in 136 tumor samples from 85 breast cancer patients. Expression of GRB7, MLN64, and p21, genes coregulated with HER2, was also quantified with quantitative RT-PCR and correlated with the overall survival (OS) and disease-free survival (DFS) individually and in combination with HER2. Results: Twenty-nine percent and 19% of the patients scored HER2 positive with IHC and quantitative RT-PCR, respectively. In 18 of 19 cases, HER2 statuses in tumors and lymph node metastases were identical. HER2 status significantly correlated with DFS when determined by IHC (P < 0.01), quantitative RT-PCR (P < 0.003), but not with FISH (P = 0.09). The combination of HER2 with MLN64, but not with GRB7 or p21, enhanced the prognostic power for the DFS (P < 0.00005) and OS (P < 0.0008). Conclusions:Quantitative RT-PCR seems to be clinically as useful in the assessment of HER2 status as IHC and FISH, yielding comparable correlations of HER2 status with the OS and DFS. Thus, quantitative RT-PCR analysis of HER2 or HER2 plus MLN64 is a promising complement or alternative to current methods for HER2 testing, particularly in laboratories lacking FISH or IHC technology. The gene for the human epidermal growth factor receptor 2 (EGFR2/erbB2/HER2/neu), hereafter referred to as HER2 , is located on the long arm of chromosome 17 (17q21.1) (1–3). HER2 encodes for a 185-kDa cytoplasmic transmembrane receptor protein (HER2-neu) with intrinsic receptor tyrosine kinase activity and 50% homology to epidermal growth factor receptor 1 (1, 4). Amplification of HER2 is among the clinically most relevant genetic aberrations in breast cancer (5). HER2 is overexpressed or amplified in 15% to 30% of all cases, which is associated with a poor prognosis, a shorter disease-free survival (DFS) and overall survival (OS), and a more aggressive cancer phenotype (5–7). It is also connected with unresponsiveness to endocrine therapy even in hormone receptor–positive patients (8, 9) and with responsiveness to anthracyclines (10–13). Furthermore, it is associated with estrogen receptor and progesterone receptor negativity, high histologic grade, high proliferation index, and a higher number of lymph node metastases (7, 10, 14). In HER2-positive patients, a combination of chemotherapy with anti-HER2 monoclonal antibodies (e.g., trastuzumab) has been shown to improve the time to progression, the response rate, and the survival rate when compared with chemotherapy alone (15). Kauraniemi et al. (16) recently found a minimal core region of amplification of about 280 kb containing not only HER2 but also GRB7, MLN64, and six further genes. GRB7 belongs to the GRB7 family of cytosolic adaptor and signaling molecules, which link tyrosine kinase receptors to downstream effectors (17). GRB7 is tightly coamplified and coexpressed with HER2 and the Imaging, Diagnosis, Prognosis Authors’ Affiliations: Departments of Obstetrics and Gynecology and Pathology, Medical University of Vienna, and Boehringer Ingelheim Austria, Vienna, Austria Received 4/15/05; revised 8/31/05; accepted 9/15/05. Grant support: Austrian Ministry of Education, Science, and Culture (Austrian Genome Research Program GEN-AU), Austrian Central Bank grant 9339, and Center of Excellence for Clinical and Experimental Oncology, Medical University of Vienna. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Martin Schreiber, Department of Obstetrics and Gynecology, Medical University of Vienna,Waehringer Guertel 18-20,Vienna A1090, Austria. Phone: 43-1-40400-2738; Fax: 43-1-40400-7842; E-mail: martin.schreiber@meduniwien.ac. at. F2005 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-0841 www.aacrjournals. org Clin Cancer Res 2005;11(23) December1, 2005 8348 Research. on July 14, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals. org Downloaded from GRB7 protein strongly binds to HER2 via its SH2 domain (18). It was suggested that the coamplification of these two genes upregulates a HER2 signaling pathway and that their coexpression is related to extramucosal tumor invasion in esophageal cancer (19). GRB7 has also been implicated in tumor progression and the regulation of cell migration (20, 21). MLN64 belongs to the START-domain family of proteins (22). Several properties of MLN64 have been ascribed to its COOH-terminal START domain, such as cholesterol binding, stimulation of movement of free cholesterol, and augmentation of steroid biosynthesis (23). Presence of excess MLN64 in breast tumors could therefore contribute to intratumoral steroidogenesis and, subsequently, to cancer progression. Like GRB7, MLN64 amplification as well as expression in human breast carcinomas closely correlates with HER2 (24). MLN64 and HER2 share similar transcriptional regulatory elements and both are activated by transcription factor SP1 (25). Genomic colocalization of nonhomologous genes that are coexpressed and involved in common biological processes has already been described in several organisms (24, 26, 27). To achieve a complex biological function, the coordinate expression of several clustered genes may be advantageous. Hence, it has been speculated that HER2, GRB7 , and MLN64, located in the same chromosomal region, are involved in a common biological pathway (25). Accordingly, aberrant activation of each of these proteins in a subset of breast tumors might contribute to their poor clinical prognosis and outcome. Overexpression of HER2 has also been shown to transcriptionally up-regulate the cyclin-dependent kinase inhibitor p21 (28). The current method of choice for HER2 testing is immunohistochemistry (IHC; refs. 29, 30), which assesses HER2 (over-) expression directly at the protein level. However, the results of IHC are not quantitative and there are no standardized scoring system and no uniformly accepted threshold for positivity. Moreover, there is a lack of standardization with regard to primary antibodies, detection systems, as well as interpretation and reporting of the results. Interpretation of IHC results is subjective and prone to interobserver variability, especially at intermediate levels of protein expression. Accordingly, it is strongly advisable to reevaluate IHC results for HER2 scored as 2+ by fluorescence in situ hybridization (FISH) because only those 2+ samples with an amplification of the HER2 gene as detected by FISH have been reported to respond to trastuzumab (31, 32). Thus, we investigated whether quantitative real-time reverse transcription-PCR (RT-PCR), an inherently quantitative method insensitive to interobserver variability and easily amenable to standardization, represents a useful alternative diagnostic approach for scoring of the HER2 status in human breast cancer. mRNA expression of HER2, GRB7, MLN64, and p21 was evaluated by quantitative RT-PCR and correlated with clinical outcome data. We show that combined scoring of HER2 and MLN64 with quantitative real-time RT-PCR outperforms the prognostic accuracy of HER2 IHC and FISH for human breast cancer.