Acute myeloid leukemia is associated with retroviral gene transfer to hematopoietic progenitor cells in a rhesus macaque by Ruth Seggewiss, Stefania Pittaluga, Rima L. Adler, F. Javier Guenaga, Cole Ferguson, Ingo H. Pilz, Byoung Ryu, Brian P. Sorrentino, W. Scott Young, Robert E. Donahue, Christof von Kalle, Arthur W. Nienhuis, and Cynthia E. Dunbar Blood Volume 107(10):3865-3867 May 15, 2006 ©2006 by American Society of Hematology

Tumor histology. Tumor histology. Myelomonocytic tumor cells with large round nuclei and a variable amount of cytoplasm infiltrate extensively in the left kidney, intercalating between renal tubular cells, shown via hematoxylin and eosin staining, original magnification, × 20 (A) and × 40 (B). Immunohistochemical stains using EnVision Plus from Dako (Carpinteria, CA) as the detection system with chromogen diaminobenzidine (DAB), showed that these cells were uniformly strongly positive for the proliferation marker MIB-1 (original magnification, × 40) (C). The cells were positive for myeloperoxidase (MPO), supporting their myeloid origin (original magnification, × 100). Images were visualized under an Olympus BX41 microscope equipped with an Olympus UPlan Fl 20 ×/0.50 (∞/0.17) (A), 40 ×/0.75 (∞/0.17) (B,C), or 100 ×/1.30 (oil) (D) objective lens (Olympus, Melville, NY). A U-TV 0.5 × C connector was used. Images were captured with an Olympus DP12 digital camera, digitally acquired with an Olympus UBS SmartMedia Reader-Writer, and processed with Photoshop 7.0 software (Adobe Systems, San Jose, CA). Ruth Seggewiss et al. Blood 2006;107:3865-3867 ©2006 by American Society of Hematology

Molecular analysis of tumor and blood. Molecular analysis of tumor and blood. (A) Southern blot analysis with an enhanced green fluorescent protein (eGFP) probe to determine copy number of the proviral genome in DNA extracted from the left kidney, which was heavily infiltrated with tumor cells. DNA (10 μg) from the tumor and from copy number control DNA obtained from Jurkat cells containing known copies per cell of the GFP gene were digested with NheI, which cuts once in each proviral LTR, and then hybridized with an eGFP probe. The copy number was calculated as 0.9 copies by phosphoimager scanning and comparison of band intensity to the Jurkat DNA copy number controls. The second panel shows 10μg tumor DNA digested with EcoR1, which cuts once within the proviral genome and therefore generates a unique band for each integration site based on the relative position of the first EcoR1 site in flanking genomic DNA. (B) Scheme of MgirL22Y retroviral vector (which contains eGFP), the internal ribosomal entry site (IRES), and the mutant dihydrofolate reductase gene (L22Y). Shown is the binding site of the probe as well as cutting sites of NheI and EcoRI. (C) BCL2-A1 is a small gene with one intron and 2 exons. The insertion occurred in the intron in the gene opposing DNA strand in chromosome band 15q25.1. Ruth Seggewiss et al. Blood 2006;107:3865-3867 ©2006 by American Society of Hematology