Background:The best current xenograft model of multiple myeloma (MM) in immune-deficient non-obese diabetic/severe-combined immunodeficient mice is costly, animal maintenance is complex and several weeks are required to establish engraftment and study drug efficacy. More practical in vivo models may reduce time and drug development cost. We recently described a rapid low-cost xenograft model of human blood malignancies in pre-immune turkey. Here, we report application of this system for studying MM growth and the preclinical assessment of anticancer therapies.Methods:Cell lines and MM patient cells were injected intravenously into embryonic veins on embryonic day 11 (E11). Engraftment of human cells in haematopoietic organs was detected by quantitative real-time polymerase chain reaction, immunohistochemistry, flow cytometry and circulating free light chain.Results:Engraftment was detected after 1 week in all embryos injected with cell lines and in 50% of those injected with patient cells. Injection of bortezomib or lenalinomide 48 h after cell injection at therapeutic levels that were not toxic to the bone marrow dramatically reduced MM engraftment.Conclusion:The turkey embryo provides a practical, xenograft system to study MM and demonstrates the utility of this model for rapid and affordable testing therapeutics in vivo. With further development, this model may enable rapid, inexpensive personalised drug screening.

4142 There are few in-vivo models for studying human leukemia and its therapy. These include the high cost immune-deficient NOD/SCID mice and large mammalian fetuses, which both require weeks to assess treatment response. We described a rapid and low-cost alternative in-vivo system for human leukemia in the preimmune chick embryo (Taizi et al, Exp Hem 34;1698-708,2006). We recently demonstrated that the turkey embryo provides a more robust model for the preclinical assessment of human leukemia infiltration (Grinberg et al, Leukemia Res 33;1417-26 2009). Here we describe the application of this powerful and inexpensive model for rapid preclinical assessment of anti-cancer therapies and basic research of blood malignancies. BM engraftment was robust occurring in 95% of the embryos using cells lines and 40% using fresh samples. Leukemia cells homed to the BM where they were already detected at 20 hours after injection and reached highest levels on days E19-24, using FACS and RT-PCR. Serial engraftment in secondary recipients of all three human stem cell leukemia lines and one fresh sample was detected in embryos injected with BM harvested 8-10 days after the first inoculation, validating the engraftment of cancer initiating cells. Human stem cell leukemia lines K562 and LAMA, (both BCR/Abl+) and CHRF (c-Kit+), and myeloma cell lines ARH-77 and CAG and fresh patient samples were injected IV into turkeys, on embryonic day E 11, using 5×106 cells from lines or 107 fresh patient cells. Engraftment of human leukemia and myeloma cells (cell lines and fresh samples) was detected 8-14 days later (E19-24), in the BM and in several hematopoietic organs at a frequency of 0.5->20%, by real-time PCR, immunohistochemistry and flow cytometry. CAG and ARH-77 myeloma cells engraftment was also detected by the presence of human monoclonal free light chain (6-10 mg/L) in blood collected from vessels of the chorioallantoic membrane, one week after cell injection. The growth of leukemias treated with doxorubicin or the tyrosine-kinase inhibitor Imatinib and myeloma with Velcade, at levels that were not toxic to the developing embryonic BM, was dramatically inhibited in vivo when the drug was injected together with cells on E11 or 48-72 hours after injection of the cells and homing to the BM. Using flow cytometry analysis the frequency of CHRF cells (detected with anti-human CD33) was reduced from 8% to 0.01% and K562 and LAMA (detected with anti-human CD71) from 1%-3% engraftment to <0.17% following treatment with Imatinib. Q-PCR analysis supported these results showing an average 8 fold reduction of CHRF and a 2-5 fold reduction of K562 and LAMA cells in the Imatinib treated turkey embryos. The ARH-77 cells (detected with anti-human CD38 and CD138) were inhibited from 8.5% to 0.72% after Velcade treatment, with a 16.5 fold reduction determined by Q-PCR analysis compare to untreated embryos. These results prove the efficacy and demonstrate the utility of the turkey embryo as a new complementary in-vivo model for studying cancer initiating cells and the growth of human blood malignancies and their response to treatment. With further improvements, it may provide an affordable, rapid in vivo system for studying the growth blood malignancies and help reduce time and cost of drug development. Disclosures: No relevant conflicts of interest to declare.

Background A widely accepted in vivo model for studying leukemia and its treatment is the highly immune-deficient mice NOD/SCID (b2M-/- or rag-/-). While this model is powerful and recapitulates the phenotypes of blood malignancies in vivo. it is costly and complex, requiring 1-2 months to establish engraftment and the mice are susceptible to spontaneous neoplasms. For these and other reasons the testing of new drugs on leukemia is primarily performed in vitro. The development of antileukemia therapies could be facilitated by a rapid and cost-effective in vivo system for evaluating human leukemia growth and its response to new drugs. Additionally, the treatment of relapsed or refractory disease could be individually tailored by this rapid and cost-effective in vivo system by evaluating patient's cells response to new agents. Turkey embryos are inexpensive, require no maintenance, are larger than chicks are more easily manipulated and have a more robust engraftment (Grinberg I, et al, Leuk Res, 2009; 33:1417-26). We recently described this new in-vivo system for studying multiple myeloma in the pre-immune turkey embryo (Farnoushi, Y., et al.,Br J Cancer, 2011; 105:1708-18). We now demonstrate application of this rapid alternative xenograft system for the preclinical assessment of leukemia growth and therapy. Methods BCR/Abl+ human leukemia lines K562 or LAMA-84 c-Kit+ CHRF 4288 and fresh patient cells were injected into turkey egg chorioallantoic membrane (CAM) veins. Cell injections were performed on day embryonic day E11as previously optimized (Farnoushi, Y., et al.,as above). To determine the engraftment of human AML cells on E19-23, in hematopoietic tissue, the engraftment of human AML cells in the BM was detected in BM by flow cytometry (FC) using anti-human CD71 for LAMA and K562, anti- human CD33 for CHRF and fresh leukemia samples. Engraftment in bone marrow (BM) and other organs was also monitored using Quantitive real time PCR (Q-PCR) comparing the amount of genomic human to the amount of avian DNA and number of human cells / avian cells in BM. Drug response was tested by IV injection of therapeutic range doses of Imatinib (Glivec ®) and Doxorubicin, 48H after cell grafting, at drug levels precalibrated to be non-toxic to the developing embryo by LD50 and BM cell viability compared to control. Six days later (E19) the embryos were sacrificed and the BM collected for FC and hematopoietic and non-hematopoietic tissues for molecular analysis. Results The optimal treatment and readout times were resolved by injecting cells on E11 and determining the kinetics of leukemia cell engraftment in the BM on E15, E18, and E23 in BM and liver. The highest engraftment level in the BM bone marrow (BM) and liver of lines tested was detected at E18 by Q-PCR, and FC in more than 90% of the injected embryos. The average engraftment (±s.d.) in the BM after one week was 4.6%+0.75 K562, 5.16%+2.15 LAMA-84, 7.65%+1.15 CHRF-4288 ( n=7-12 per group) and 2.5% fresh leukemia cells was detected by FC. Q-PCR results were similar to those of FC. Imatinib toxicity testing revealed 100% survival of embryos with no BM toxicity on embryos treated on E13 with doses similar to a human therapeutic dose, up to 0.75 mg/egg. Treatment of embryos with 100 ug Doxorubicin was previously shown to be not toxic to the embryos (Taizi M et al. Exp Hematol 2006; 34:1698–708). A single dose of 0.75 mg Imatinib/embryo dramatically reduced engraftment in BM and several other organs of all 3 AML cell lines or fresh patient leukemia cells. A similar effect was also obtained by a single dose Rx 100ug Doxorubicin. Treatment of a single dose of 0.75 Imatinib mg/embryo 48H after injecting ARH-77 (multiple myeloma) had no effect on cell engraftment. Treatment with a single non toxic dose of Revlimid as previously described (Farnoushi, Y., et al. as above) eliminated engraftment of ARH77 cells, clearly demonstrating the specificity of the drug treatments. Conclusions The results presented demonstrate the potential utility of a practical avian embryo model for testing drug activity in vivo. With further work the turkey embryo may provide a new xenograft in vivo method for studying the biology of leukemia engraftment, and for rapidly and affordably testing leukemia therapies. This system may provide a new platform for developing individualized patient screening for response or resistance to particular therapeutic agents. Disclosures: No relevant conflicts of interest to declare.