387. Myeloid Sarcoma Associated with Gene Marking of Hematopoietic Stem Cells in a Rhesus Macaque
Authors: Ruth Seggewiss, Stefania Pittaluga, Rima Adler, Cole Ferguson, Elio F. Vanin, Patrick F. Kelly, Robert E. Donahue, Brian P. Sorrentino, Arthur W. Nienhuis, Cynthia E. Dunbar
Journal: Molecular Therapy (2005)

Replication defective retroviruses are attractive tools for hematopoietic stem cell (HSC) gene therapy because of their ability to integrate into genomic DNA. Reports of neoplasia related to vector insertional proto-oncogene activation have raised safety concerns, however in these instances the vector itself carried a potentially cooperating transforming gene. We report for the first time a replication-defective vector-associated neoplasia in a non-human primate. Six years after autologous transplantation with G-CSF/SCF mobilized CD34+ cells transduced with a MSCV RD114 pseudotyped retroviral vector expressing eGFP and a drug resistant variant of the human dihydrofolate reductase gene (L22Y), a rhesus macaque developed a fatal myeloid sarcoma. Of note, this animal had high level transient marking during the first year following transplantation derived from a single transduced clone containing two vector insertions, over the next five years 1-3% GFP+ blood cells and was clinically well (Kelly et al, 2003). The animal received one cycle of trimextrexate and NBMP with transient increase in GFP+ blood cells 2001 (Persons et al, 2004). In 9/04 the animal developed abdominal distress and had a renal mass. Blood showed 30% GFP+ granulocytes, but no leukemia. Following surgery the animal died of pancreatitis and sepsis. Necroscopy revealed myelomonocytic tumor cell infiltrates with large round nuclei and variable amount of cytoplasm (MPO+, CD45+/-, CD68+, CD3-, CD20-, -/-, GFP+/-) in both kidneys, liver, pancreas, spleen, lymph nodes and in the choroid plexus. Taqman demonstrated a vector copy number of at least 1/cell in the tumor, suggesting association with vector. There was no detectable replication-competent helper virus in the tumor or the blood, based on PCR for helper gag/pol sequences. Blood cells and enriched tumor cells (>95%) have been analyzed via LAM-PCR identification of integration sites. A vector insertion in the first intron of the BCL2-A1 gene was found in the tumor, along with a second valid integration site that could not be mapped unequivocally using the human genome. PCR analysis using primers specific for the BCL2-A1 insertion suggested very high level contribution from the clone in the first year following transplantation, which then became undetectable and increased in the blood concurrent with the development of the myeloid sarcoma. Thus a single clone containing the vector insertion into the BCL2-A1 gene made a major multilineage contribution to hematopoieses during the first six months after reconstitution, became dormant and then re-emerged as a myeloid neoplasm after six years. The BCL2-A1 protein blocks apoptosis and is associated with a poor prognosis in human acute myeloid leukemia. Further research is required for risk assessment of current available vector systems, especially when targeting long-lived HSC.