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Surviving leukemic stem cells (LSCs) after chemotherapy lead to relapses in acute myeloid leukemia (AML). Because LSCs will not be eradicated after standard chemotherapy, inhibiting the propagation of AML cells by LSCs is a major part in AML treatment. Although CD34+CD38- cells in leukemia are representative LSCs, it is still difficult to distinguish them apart from normal hematopoietic stem cells (HSCs). So far, many studies have shown rapid advances in phenotypic characterization. However, heterogeneous diversity in AML patients may not allow effective eradication of LSCs. Vascular endothelial growth factor receptors (VEGFR)-3 is expressed in AML blasts and in the bone marrow (BM) environment including sinusoidal vessels. In particular, VEGFR-3 is strongly correlated with poor prognosis, leukemic cell proliferation and survival in AML. Based on previous reports, we were able to hypothesize that VEGFR-3 is expressed in LSCs and functions as a LSC marker. Here, we observed high expressions of VEGFR-3 on CD34+CD38− cells in AML patients who received chemotherapy and further showed low homing and engraftment capacity of CD45dim blast cells in the BM by a VEGFR-3 antagonist. In order to determine the expression of VEGFR-3 on LSCs, data was collected from 64 AML patients, 12 of whom were after complete remission (CR), as well as from 14 healthy volunteers. MNCs were first isolated and were then subjected to FACS analysis and immunocytochemistry. NOD-Scid IL2RγNull mice were used for homing efficiency and engraftment in vivo. MAZ51 was used as a VEGFR-3 antagonist. FACS analysis showed that VEGFR-3 was increased on CD34+CD38− LSC cells. (Normal vs. AML vs. CR, VEGFR-3 on CD34+CD38- cells: 8.33 ± 4.37% vs. 25.62 ± 2.46% vs. 23.46 ± 5.47%, P < 0.05). Similarly, immunocytochemistry clearly displayed the co-expression of VEGFR-3 on isolated CD34+CD38− LSC cells, suggesting the possibility of it as a LSC marker. We checked the ability of LSCs to use colony forming units assay. VEGFR-3+CD34+ cells showed unarguably enhanced colony forming ability compared to that of VEGFR-3-CD34+ cells from patients. To test whether VEGFR-3-CD34+ cells are not on apoptotic procedure, annexin-V and proliferation assay with Ki67 were performed, and there was no difference in apoptotic and proliferative movement in both cells. Intending to determine homing efficiency and engraftment, CD34 and CD45 markers were used in the BM and it was discovered that sorted VEGFR-3+CD34+CD38- cells showed significantly increased homing and engraftment efficiency compared to those of VEGFR-3-CD34+CD38- cells (by FACS, homing capacity: 1.93 ± 0.19% vs. 0.13 ± 0.06%, P = 0.02; engraftment: 26.4 ± 9.42% vs. 5.30 ± 2.31%, P < 0.05), implying that VEGFR-3 can serve as a marker for LSCs. We demonstrated that VEGFR-3 was highly expressed and enriched on CD34+CD38- cells in CR status as well as in the initial diagnosis of AML. Therefore, the targeting of VEGFR-3 may diminish LSC function in human AML. These findings could suggest some clues to develop therapeutic strategies targeting VEGFR-3+ LSCs with favorable tumor microenvironments. Disclosures No relevant conflicts of interest to declare.

Abstract 4953 Introduction: Although treatment with hypomethylating agents such as azacitidine or decitabine has been the standard of care for patient with myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML), about half of the patients fail to respond to the agents and most responders progress within 2 years. Retrospective studies showed poor outcomes after failure of treatment with azacitidine or decitabine and there is no standard salvage therapy for patients who fail hypomethylating treatment (HMT). We retrospectively studied outcomes of patients who failed HMT and analyzed the effects of salvage therapy after HMT failure. Methods: Between September 2006 and October 2010, a total of 149 patients were treated with either azacitidine (n=75) or decitabine (n=74) for MDS defined by the WHO classification and chronic myelomonocytic leukemia (CMML) in 3 Korean institutes. Ninety-one of the 149 patients were included in this study and disease status at the end of HMT was categorized as stable disease (n=22), primary progression (n=17), progression after response (n=38), and intolerance (n=14). Six patients who were still receiving hypomethylating agents with a median of 19 courses (range, 15 to 48) and 52 patients who stopped hypomethylating agents for other reasons were excluded from the analysis. Results: Median age was 59 years (range, 23 to 80) at the time of HMT failure. Median follow-up duration of surviving patients was 47. 8 months (range, 5. 8 to 62. 9) and 69 patients died. Probability of overall survival (OS) at 3 years was 28. 1% and median OS was 12. 1 months (95% confidence interval [CI], 9. 8 to 14. 4). Multivariate analysis showed that disease status and evolution to acute myeloid leukemia (AML) at HMT failure were independent prognostic factors for OS. A total of 37 patients (40. 7%) received supportive care only after HMT failure and other patients were managed with one or more treatments including immunosuppressive therapy (n=7), low-dose cytarabine (n=9), androgen (n=8), alternate azanucleoside (n=2), intensive chemotherapy (n=24), and allogeneic hematopoietic cell transplantation (HCT) (n=23). Objective response to non-transplant treatment was observed in 11–17% of evaluable patients, while 17 (74%) of 23 patients who received allogeneic HCT attained complete response. Probability of OS at 2 years (from HCT) was 60. 9% in the transplanted patients; it was 78. 6% in patients who received HCT during MDS and 33. 3% in those who received HCT after AML evolution (P=0. 016). Conclusions: The clinical outcomes of patients after hypomethylating treatment failure are poor; especially, AML evolution at the time of hypomethylating treatment failure and primary progression after hypomethylating treatment indicated very poor prognosis. Responses to various low intensity therapies and intensive chemotherapy were infrequent. Long-term survival without disease evidence was observed in about half of the patients who received allogeneic HCT. In appropriately selected patients, allogeneic HCT should be performed in earlier period, especially before evolution to AML. Patients with MDS that has failed to respond to hypomethylating agents should be referred for clinical trials when available. Disclosures: No relevant conflicts of interest to declare.