Human cell lines of putative HCL origin lack the BRAF-V600E mutation and key phenotypic features of HCL. Direct DNA Sanger sequencing of BRAF-exon15⁴  in the HC-1 cell line (right chromatogram) shows the absence of the T-to-A point mutation at codon 600 leading to the V600E amino acid replacement, which is instead present heterozygously in primary leukemic cells MACS-purified from the peripheral blood of an HCL patient (left chromatogram). Both HC-1 cells and patient's leukemic cells display a wild-type codon 594 (GAT), as opposed to HAIR-M cells harboring a clonal heterozygous T-to-A point mutation at this codon (not shown) that leads to the D594E amino acid replacement. HC-1 cells xenografted in an NSG mouse show diffuse infiltration of the spleen by large B-cell lymphoma-like cells (right H&E staining) that are negative for annexin-1 (ANXA1; right ANXA1 immunostaining¹ ). Conversely, the splenectomy specimen of the HCL patient is infiltrated by small mature-looking lymphoid cells with wide pale cytoplasm (left H&E staining) strongly expressing annexin-1 (left ANXA1-staining). All micrographs were collected using an Olympus B61 microscope (equipped with an Olympus UPlanApo 40×/0.8 NA objective and with an Olympus E330-ADU1.2x camera) and were acquired and processed using Olympus cell∧B imaging software.