Identification and validation of serum biomarkers for clinical assessment of Hsp90 inhibitor therapy

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Background:
 Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a crucial role in the conformational maturation of numerous oncogenic signaling proteins. Tumor cells are known to express an altered high affinity conformation of Hsp90 to accelerate the folding of proteins and support their rapid growth. We developed fully synthetic small molecules (e.g., BIIB021) that preferentially bind to the tumor specific Hsp90 and inhibit cancer cell growth in a variety of xenograft models. Several Hsp90 inhibitors including BIIB021 are currently being tested in human cancer patients. The pharmacodynamic (PD) activity of Hsp90 inhibitors is presently assessed by a Western blot assay that is both semi-quantitative and difficult to implement in the clinic. We identified and clinically validated three serum biomarkers (e.g., Hsp70, Hsp90α, HER-2 ECD) via a bioinformatics approach as well as traditional methods. Assessment of these serum biomarkers is easy to implement and should facilitate a systematic evaluation of Hsp90 inhibitor therapy in the clinic and could help determine optimal biological dose, selection of formulations and aid in determination of pharmacologically active dose for advanced Phase II clinical trials.
 Methods:
 Tumor cell lines (e.g., H146 and BT474) and human PBMC were treated with various doses of novel synthetic Hsp90 inhibitors, BIIB021 and BIIB028. Proteins secreted into the culture medium or interstitial medium (Xenograft models) at various intervals were analyzed by a bioinformatics approach (non labeled multidimensional separation of peptide digests coupled with ESI-MS), Western blotting or selected biomarkers by highly sensitive ELISA methods both on chemiluminescence and colorimetry platforms. Based on these preliminary analyses, at least three secreted pharmacodynamic markers viz., Hsp70, Hsp90α and HER-2ECD were further validated for clinical use and parameters including accuracy, precision, matrix effects and stability were assessed. Additionally, the basal levels of all three PD markers in normal human serum and multiple different cancer patient serum were analyzed and satisfactorily detected within the assay range. Furthermore, the assays for serum Hsp70 and HER-2ECD have been successfully implemented for the analysis of biomarkers in serum from cancer patients undergoing treatment with BIIB021 in Phase I solid tumor trials. 
 Results and Conclusions:
 Systematic and simpler approaches to monitor the PD activity of Hsp90 inhibitors in the clinic are currently lacking. We report here the identification, development and validation of clinical biomarkers to monitor the bioactivity of Hsp90 inhibitors in early stage clinical trials. These validated assays should be useful in selection of appropriate formulations, dose and monitoring the biological effects of investigational Hsp90 inhibitors in cancer patient trials.