Differences in the Angiogenic Response and Subsequent Growth of Plasma Cells From Myeloma and MGUS Patients Xenografted Into Zebrafish Embryos.

Abstract 2912 A monoclonal gammopathy of uncertain significance (MGUS) is an obligate precursor to each case of multiple myeloma (MM). MGUS and MM samples share many of the same gene mutations and chromosomal translocations. Despite increasing genetic and genomic information gathered from patient samples, we still do not understand which mutations are responsible for the progression from MGUS to MM. In order to learn more about this progression and the possible involvement of an angiogenic switch as seen in solid tumors, we have developed a zebrafish model to study the angiogenic potential and subsequent growth of CD138+ plasma cells derived from normal human bone marrow, MGUS and myeloma patients. Immunopurified CD138+ cells were labeled with the red fluorescent dye CM DiI, suspended in Matrigelâ and injected into the perivitelline space of Tg(fli1:GFP) zebrafish embryos that had fluorescent vasculature, 48 hpf (hour-post-fertilization). Cell growth and angiogenesis were assessed by conventional and confocal fluorescent microscopy. Inhibitors of myeloma cell growth or tumor-induced angiogenesis were added to embryo water. The injection of up to 2000 plasma cells from normal human bone marrow into zebrafish embryos did not elicit an angiogenic response and the cells did not grow after injection. In contrast, the injection of as few as 100 plasma cells purified from the marrow of patients with multiple myeloma induced a brisk angiogenic response with extension of new vessels ventrally from the sub-intestinal venous system into the site of cell injection. Angiogenesis was accompanied by the growth of the CD138+ cells and, in some cases, movement of cells out of the Matrigelâ plug to distant sites in the developing embryo. MGUS cells induced minimal or no angiogenesis and grew very slowly after injection. The growth of cells from MGUS patients and de novo myeloma patients was inhibited by the addition of the VEGFR2 inhibitor SU5416 to embryo water 24 hpi (hours-post-tumor cell-injection). If the addition of SU5416 was delayed until 48 hpi, when the angiogenic response had been established, there was no effect on cell growth. We also observed that dexamethasone, bortezimib and lenalidomide, singly and in combination, inhibited the growth of MGUS and myeloma cells. These studies document clear biologic differences between normal, MGUS and myeloma plasma cells. In addition, we provide evidence that the zebrafish embryo can be used to study tumor-induced angiogenesis, tumor cell growth and the effects of possible inhibitors. We also provide some new insights into the mechanism underlying the disappointing response of angiogenic inhibitors when given to patients with multiple myeloma. Disclosures: Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

CD38 Status of CD34+ Myeloblasts, but Not Side Population (SP) Frequency, Predicts Initial Response to Induction Therapy In Patients with Newly Diagnosed Acute Myeloid Leukemia (AML).

Abstract 1056 Objective: Evaluate prognostic significance of immunophenotypically and functionally defined candidate leukemia stem cell populations. Background: Numerous studies have demonstrated that the population of CD34+/CD38dim (low to negative) cells is highly enriched for human hematopoietic stem cells. Another marker of stem cell-like phenotype can be assessed functionally by measuring the ability of the blasts to actively export DNA binding dye, Hoechst 33342 by flow cytometry giving rise to the so called “side population”. SP has also been widely linked to stem cell potential in both hematopoietic and non-hemtatopoietic tissues. The relationship between SP and CD34+/CD38dim phenotype is not entirely clear. Analogously, most human acute myeloid leukemias contain both a CD34+/ CD38dim population and a side population. CD34+/CD38dim population has been established as a source of AML cells with high self renewal potential. Less data exists for the role of SP in self renewal in AML. The relationship between the CD34+/CD38dim population and the side populations in AML has not been fully clarified. Methods: Twenty-two newly diagnosed AML patients were identified with CD34+ immunophenotype on the majority of leukemic blasts amongst patients enrolled on an IRB-approved protocol for analysis of blood and bone marrow samples. These patients then underwent induction chemotherapy, and response and duration of first complete remission (CR1) were determined. Wilcoxon rank sum test was used to compare stem cell populations with achievement of CR and FLT3 status; Kruskal-Wallis test to compare stem cell populations across cytogenetic categories and CR1 duration categories (more then 12 months, relapsed less than 12 months, no response), and Spearman correlation to assess correlation between age and stem cell population. For the purposes of analysis of AML prognosis fractions of blasts with CD38 expression below that of mature myeloid cells (CD38low) and those with no CD38 expression (CD38neg) expression were analyzed separately. Results: The median age of AML patients was 61 (range 19–84). 23% of the patients had favorable, 41% intermediate, and 36% poor risk cytogenetics 45% achieved complete remission. Median SP % was 0.04 (0-13.2), median CD38low % was 1.298 (0.02-17.4), and median CD38neg % was 0.01077 (0-0.92). We first analyzed the relationship between CD34+/CD38dim and SP phenotypes in normal and AML patients. 20 normal human bone marrow and 22 bone marrows from patients were investigated by flow cytometry. We demonstrate that normal human bone marrow SP is highly enriched for the most phenotypically immature CD34+/CD38dim small blasts likely representing the earliest identifiable hematopoietic precursors. Moreover, the highest dye efflux activity correlated with the least CD38. In contrast to normal marrow in AML, the SP did not correspond with CD38 expression levels. Most samples showed an SP phenotype that extended to include many CD38bright events more typical of lineage-committed progenitors. ABCG2 pump inhibitor Fumitremorgin C blocked dye efflux in all normal and all but one AML samples we tested. Side population did harbor the same cytogentic anbormalities as the bulk of blasts in 4/4 samples tested. SP percent did not correlate with age, cytogenetic risk category, response, CR1 duration, or Flt3 (all p>0.05). Percent of CD38low and CD38neg each negatively correlated with achievement of complete response to treatment (both p<0.01). Percent CD38neg correlated with length of CR1 (p=0.02). There was also a positive correlation with increasing age (both p=0.01) for both CD38low and CD38neg populations. Only percent CD38neg correlated with cytogenetic risk category (p=0.03). Conclusions: Expansion of less mature (CD38 lower) compartment of blasts may explain the adverse prognosis of patients with advanced age and poor cytogenetic predictors. Disclosures: Becker: Genzyme: Research Funding.