Molecular drivers underlying bone metastases in human cancer are not well understood, in part due to constraints in bone tissue sampling. Here, we undertook RNA sequencing of circulating tumor cells (CTCs) obtained from blood samples of women with metastatic estrogen receptor (ER)+ breast cancer, comparing cases with progression in bone versus visceral organs. Among the activated cellular pathways in CTCs from bone-predominant breast cancer is Androgen Receptor (AR) signaling. AR gene expression is evident, as is its constitutively active splicing variant AR-v7. AR expression within CTCs is correlated with the duration of treatment with aromatase inhibitors, suggesting that it may contribute to acquired resistance to anti-estrogen therapy. In an established breast cancer xenograft model, a bone-tropic derivative displays increased AR expression, whose genetic or pharmacologic suppression reduces metastases to bone but not to lungs. Together, these observations identify AR signaling in CTCs from women with bone-predominant ER+ breast cancer, and provide a rationale for testing androgen inhibitors in this subset of patients.
Overall design: Thirty-two women with metastatic ER-positive breast cancer were enrolled in an IRB-approved observational study at Massachusetts General Hospital (MGH). All patients had clinical evidence of disease progression, and had discontinued their therapy at the time of CTC collection, in anticipation of starting an experimental early phase drug trial (“drug washout period”). Patients with detectable CTCs (n=22) were classified dynamically based on which metastatic sites were progressing at the time of blood sampling, identifying 13 (59%) who had the appearance of new bone metastases at the time of CTC isolation (termed “Bone (+)”), versus 9 (41%) who had either no bone involvement at all or had no new bone metastases, despite having disease progression at other soft tissue or visceral sites (termed “Visceral (+)”). For each patient, approximately 10ml of blood was processed through the CTC-iChip, which first uses microfluidic size-based fractionation to separate nucleated cells from platelets and red blood cells (RBCs), and then effectively depletes magnetically-labeled white blood cells (WBCs) from untagged CTCs. From the initial sample of blood (10 x 106 WBC/ml), the final microfluidic product contains approximately 500 residual WBCs per ml of whole blood processed, along with candidate CTCs. Unfixed CTCs are identified by live staining with Alexa 488-conjugated antibodies against the cell surface markers EpCAM (epithelial-specific), HER2 (lineage-specific) and CDH11 (mesenchymal-specific), while contaminating WBCs are counterstained using TexasRed-conjugated antibodies against the hematopoietic lineage-specific markers CD45, CD16 and CD14. Marker-positive single and clustered CTCs were identified in 13 patients with Bone (+) and 9 patients with Visceral (+) disease, with a median of 3.5 CTCs per patient, leading to a total of 77 samples representing either single CTC, CTC-clusters, or in rare patients with very high numbers of CTCs, pools of up to three CTCs. RNA from these CTCs was then extracted, reverse transcribed, amplified and sequenced as described in PubMed ID 25171411.
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