Abstract 209: Proteomic analysis of therapeutic biomarkers in decalcified bone metastases
Authors: Cecchi Fabiola , Sellappan Shankar , Schwartz Sarit , Gong Chao , Darfler Marlene , Scott Kerry , Mamus Steven W. , Emmert-Buck Mike , Hembrough Todd 
Published: July 2017

Abstract
Introduction: Targeted therapies successful in treating primary tumors are often ineffective against corresponding metastatic lesions. Tumors and their bone metastases are biologically distinct, as reflected by discordant biomarker status. Decalcification of bone destroys protein and can preclude molecular analysis. A profiling method that does not depend on tissue antigenicity or nucleic acid preservation could increase the diagnostic value of decalcified bone samples. We assessed the effects of decalcification on proteomic analysis of tumor tissue. We also quantified 27 therapeutically-relevant proteins in decalcified bone metastases of cancer patients using mass spectrometry-based proteomic analysis.
Methods: To examine the effects of decalcification on protein quantification, we used 3 non-bone tissue specimens from lung adenocarcinoma, lung squamous cell carcinoma, and colon medullary carcinoma. Non-bone tumor tissue was expected to perform similarly in proteomic analyses to bone tissue, which was not available. Tissue specimens were processed without decalcification and with hydrochloric acid-based Decal-Stat™ decalcification solution for 1, 3, 12, and 24 hours prior to paraffin embedding, tissue sectioning, and mass spectrometric analysis. Proteomic analysis was also performed on 26 previously decalcified biopsies of metastatic bone lesions from patients with cancers of the lung (n=7), breast (n=7), stomach (n=3) and 8 other indications. Archived tumor tissue was microdissected and solubilized to tryptic peptides. Target proteins in each liquefied tumor sample were quantitated in triplicate with a multiplexed, selected reaction monitoring mass spectrometry assay.
Results: In the non-bone tumors, non-decalcified & decalcified samples yielded similar quantities of total protein (range: 19.2 - 24.1 µg) and of all 20 protein biomarkers detected. The 26 bone metastases expressed 20 of the 27 protein targets tested. Of the 7 bone samples from lung cancer patients, 5 expressed EGFR protein and 5 expressed hENT1 protein (a marker of response to gemcitabine). A metastatic bone tumor from a breast cancer patient expressed hENT1 (129 amol/µg) and overexpressed HER2 (5750 amol/µg; this exceeds a level previously correlated with increased survival in trastuzumab-treated patients). Bone lesions from prostate & gynecologic cancers overexpressed AR, hENT1, EGFR and TOPO1 proteins.
Conclusions: A commonly used decalcifying solution had no discernable effects on proteomic quantification of biomarker proteins in archived tumor samples. Targeted proteomics can quantify an entire panel of therapeutically-relevant proteins from a single decalcified bone biopsy specimen. Proteomic analysis of bone metastases upon diagnosis of metastasis or at relapse could inform treatment decisions, particularly in patients who have disease progression only in bone lesions or whose bone biomarkers are discordant from those of the primary tumor.
Citation Format: Fabiola Cecchi, Shankar Sellappan, Sarit Schwartz, Chao Gong, Marlene Darfler, Kerry Scott, Steven W. Mamus, Mike Emmert-Buck, Todd Hembrough. Proteomic analysis of therapeutic biomarkers in decalcified bone metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 209. doi:10.1158/1538-7445.AM2017-209