Immunohistochemistry

Formalin-fixed paraffin-embedded (FFPE) tissue blocks from primary and metastatic clear cell RCC (ccRCC) were retrieved from Beth Israel Deaconess Medical Center under Dana Farber/ HCC IRB approved protocol 01-130. 12 primary ccRCCs were retrieved and one primary ccRCC was paired with a corresponding lung metastasis. Analysis was performed in selected areas of primary tumors containing low (G1-G2) and high (G3-G4) Fuhrman nuclear grade (FNG). Immunohistochemistry was performed on four micron-thick, FFPE tumor sections, which were initially deparaffinized, rehydrated and heated with a pressure cooker to 125°C for 30 seconds in citrate buffer for antigen retrieval and then incubated with peroxidase (Dako #S2003, Carpinteria, CA) and protein blocking reagents (Dako #X0909) respectively for 5 minutes. Sections were then incubated with anti-BMP-9 antibody (AbD Serotec #1406-1460) at 1:2000 dilution for 1 hour at room temperature followed by incubation with the Dako EnVision+ System HRP labeled polymer anti-rabbit (Dako #K4011) for 30 minutes. All sections were developed using the DAB chromogen kit (Dako K3468) for 2 minutes and then lightly counterstained with hematoxylin. The assay was validated using CHO cells that over express human BMP9 (generated at Acceleron Pharma, Inc.) and the parental CHO cell line. The presence of tumor cells with cytoplasmic staining was assessed. A case was considered positive if any positivity was detected. Immunohistochemistry of CD34 was performed on adjacent four-micron thick FFPE sections from sunitinib and ACE041, sunitinib only and vehicle treated xenografts as previously [37].