BRAF inhibition downregulates the expression of HCL-specific markers in vivo. (A) Immunohistochemistry shows strong cyclin D1 downregulation (brown nuclear staining) by HCL cells (defined by the blue membrane staining for CD20) in bone marrow biopsies of a trial HCL patient taken before and after 2 weeks of oral treatment with vemurafenib 960 mg twice daily. The arrow in the right panel indicates a cyclin D1+ non-B cell as internal control. (B) Western blotting for cyclin D1, phospho-ERK, and (as loading control) tubulin β in primary leukemic cells purified from the blood of a HCL patient (HCL 1) before and after 2 and 3 days of oral vemurafenib. A mantle cell lymphoma cell line (Jeko-1) and primary purified leukemic cells from a HCL-like patient were used as positive and negative control for cyclin D1 expression, respectively. (C) Flow cytometric expression of surface CD25 in blood leukemic cells (coexpressing CD19 and CD103; blue events in the CD45⁺ gate) of HCL patient 4 before and after treatment with oral vemurafenib for 7 and 14 days. Red events represent the rest of CD45⁺ blood cells.