Assessing efficacy of novel Hsp90 inhibitors in breast cancer patients by enumeration of circulating tumor cells and HER-2 degradation

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Background:
 Hsp90 inhibitors are currently under investigation in multiple human (Phase I-III) cancer trials. We have a fully synthetic Hsp90 inhibitor (BIIB021) in development that effectively inhibits tumor growth in a variety of tumor models. BIIB021 is being investigated for the treatment of solid tumors and metastatic breast cancer in Phase I clinical trials. To evaluate the effect of BIIB021 on HER-2 degradation in clinical trials, we used a systematic approach to develop a semi-quantitative method for estimating HER-2 expression on circulating tumor cells (CTC) utilizing the Veridex technology. As a first step, we used control breast cancer cell lines with varied HER-2 expression levels to establish a scoring system to predict changes in HER-2 receptor (intensity) levels. Additionally, we treated these control cells with different doses of BIIB021 to demonstrate HER-2 degradation on CTC. This assay will be used for assessing the pharmacological activity of BIIB021 in the ongoing metastatic breast cancer patient trials.
 Methods:
 CTC analysis was performed on the Immunicon/Veridex cell tracks system using the CTC IVD kit coupled with HER-2/neu immunophenotyping reagent. To establish a scoring system (0 to 3+) for predicting changes in HER-2 expression levels, the receptor intensities of four breast cancer lines (BT474, MDA-MB-361, MDA-MB-175, MCF7 having high to very low HER-2 receptor numbers) were spiked into human blood and analyzed using the HER-2 CTC system. Next, breast cancer cells expressing high (BT474) and low (MCF7) levels of HER-2 receptor were treated with a titrated dose of BIIB021 and HER-2 expression levels determined by flow cytometry. Based on the flow cytometry data, BT474 (high Her2-expression), MDA-361, MDA-175 (intermediate HER-2 expressor), and MCF7 (low Her2 -expression) cells were treated with BIIB021 for 16 hours and changes in HER-2 expression levels were assessed on the CTC platform using the preestablished scoring system.
 Results/Conclusions: We established a receptor intensity chart for scoring HER-2 levels on CTC following Hsp90 inhibitor therapy. Based on this scoring system over 80% of the MDA-MB-361 and 40% of MDA-MB-175 cells treated with BIIB021 showed complete HER-2 degradation (scored ‘0’) and a majority (>90%) of the BT474 cells had low levels (1+) of HER-2. This semi-quantitative scoring system should be useful in assessing the pharmacological activity of Hsp90 inhibitors in breast cancer patients with high to intermediate HER-2 expression levels and coupled with the ability to enumerate changes in absolute number of metastatic cancer cells, the HER-2 CTC platform should provide a powerful way to demonstrate the pharmacological efficacy of Hsp90 inhibitors in metastatic breast cancer patients.