Purpose Flow cytometry and RT-PCR can detect occult Ewing sarcoma (ES) cells in the blood and bone marrow (BM). These techniques were used to evaluate the prognostic significance of micrometastatic disease in ES. Experimental Design Newly diagnosed patients with ES were enrolled on two prospective multi-center studies. In the flow cytometry cohort, patients were defined as “positive” for BM micrometastatic disease if their CD99+/CD45− values were above the upper limit in 22 control patients. In the PCR cohort, RT-PCR on blood or BM samples classified the patients as “positive” or “negative” for EWSR1/FLI1 translocations. The association between micrometastatic disease burden with clinical features and outcome was assessed. Co-expression of IGF-1R on detected tumor cells was performed in a subset of flow cytometry samples. Results The median total BM CD99+CD45− percent was 0.0012% (range 0–1.10%) in the flow cytometry cohort, with 14/109 (12.8%) of ES patients defined as “positive.” In the PCR cohort, 19.6% (44/225) patients were “positive” for any EWSR1/FLI1 translocation in blood or BM. There were no differences in baseline clinical features or event-free or overall survival between patients classified as “positive” vs. “negative” by either method. CD99+CD45− cells had significantly higher IGF-1R expression compared to CD45+ hematopoietic cells (mean geometric mean fluorescence intensity 982.7 vs. 190.9; p<0.001). Conclusion The detection of micrometastatic disease at initial diagnosis by flow cytometry or RT-PCR is not associated with outcome in newly diagnosed patients with ES. Flow cytometry provides a tool to characterize occult micrometastatic tumor cells for proteins of interest.

Dendritic cells (DCs) are critical for initiation of adaptive immune responses, but they also play important roles in self-tolerance, and in tumor bearing hosts, some DC subsets may contribute to immune evasion. Myeloid derived suppressor cells (MDSC) are also important mediators of tumor immune evasion. The relationship between myeloid DC and MDSCs in humans remains unclear. Human myeloid DCs are defined as CD11c+CD123-, although some have reported dim CD123 expression on human myeloid DCs. We describe a CD11c+CD123+ myeloid DC subset that shows characteristics of a MDSC. Monocytes elutriated from normal donors were incubated for 48hr with IL4 ± IL13 ± IL3. Monocytes cultured with all three cytokines acquired strong expression of both CD123 and CD11c, with 79.5% of cells CD123+CD11c+. CD11c+CD123+ cells demonstrated over-expression of both arginase and iNOS by flow cytometry and/or real-time PCR in comparison to myeloid derived DCs generated with IL4, GM-CSF and LPS (mean fluorescence intensity of arginase in the IL4, IL13 and IL3 generated cells of 5973 vs. 1961 in IL4, GM-CSF and LPS generated cells). CD11c+CD123+ DCs also coexpress IL-4Rα and IL7Rα, characteristics of MDSC and regulatory DC subsets respectively. IL3 appears to be a potent inducer of CD123 expression on CD11c+ DCs, since myeloid DCs generated with only IL4 and IL13, in combination or separately, showed a less pronounced phenotype (38-52.7% CD11c+CD123+). Moreover, CD123 expression on myeloid DCs appears to be a marker for arginase production since CD123+ cells within the IL4, IL13 and IL3 cultures show higher arginase expression than CD123- cells within the same cultures. Functional studies are ongoing. In conclusion, human myeloid DCs generated by incubating monocytes with IL4, IL13 and IL3 have a novel CD11c+CD123+ phenotype and show characteristics of myeloid derived suppressor cells, raising the prospect that IL3 plays an important role in generating myeloid derived suppressor cells in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3818.

Hepatosplenic gamma-delta T-cell lymphoma is a very rare, aggressive form of peripheral lymphoma first recognized in 1990. Patients often present with organomegaly, anemia, adenopathy, and B symptoms. Rarely in the literature is a pediatric patient described with this subtype of peripheral T-cell lymphoma. Also, retinal hemorrhages have never been described as a presenting symptom of hepatosplenic gamma-delta T-cell lymphoma. We describe an adolescent patient with hepatosplenic gamma-delta T-cell lymphoma who presented with retinal hemorrhages, massive splenomegaly, bone marrow involvement, and B symptoms.

<div>Abstract<p><b>Purpose:</b> Flow cytometry and RT-PCR can detect occult Ewing sarcoma cells in the blood and bone marrow. These techniques were used to evaluate the prognostic significance of micrometastatic disease in Ewing sarcoma.</p><p><b>Experimental Design:</b> Newly diagnosed patients with Ewing sarcoma were enrolled on two prospective multicenter studies. In the flow cytometry cohort, patients were defined as “positive” for bone marrow micrometastatic disease if their CD99⁺/CD45⁻ values were above the upper limit in 22 control patients. In the PCR cohort, RT-PCR on blood or bone marrow samples classified the patients as “positive” or “negative” for <i>EWSR1/FLI1</i> translocations. The association between micrometastatic disease burden with clinical features and outcome was assessed. Coexpression of insulin-like growth factor-1 receptor (IGF-1R) on detected tumor cells was performed in a subset of flow cytometry samples.</p><p><b>Results:</b> The median total bone marrow CD99⁺CD45⁻ percent was 0.0012% (range 0%–1.10%) in the flow cytometry cohort, with 14 of 109 (12.8%) of Ewing sarcoma patients defined as “positive.” In the PCR cohort, 19.6% (44/225) patients were “positive” for any <i>EWSR1/FLI1</i> translocation in blood or bone marrow. There were no differences in baseline clinical features or event-free or overall survival between patients classified as “positive” versus “negative” by either method. CD99⁺CD45⁻ cells had significantly higher IGF-1R expression compared with CD45⁺ hematopoietic cells (mean geometric mean fluorescence intensity 982.7 vs. 190.9; <i>P</i> < 0.001).</p><p><b>Conclusions:</b> The detection of micrometastatic disease at initial diagnosis by flow cytometry or RT-PCR is not associated with outcome in newly diagnosed patients with Ewing sarcoma. Flow cytometry provides a tool to characterize occult micrometastatic tumor cells for proteins of interest. <i>Clin Cancer Res; 22(14); 3643–50. ©2016 AACR</i>.</p></div>

<p>Supplemental Table 1. Association of clinical features with BM micrometastatic cell burden (CD99+CD45- percent) in flow cytometry cohort. Supplemental Table 2. Total CD99+CD45- percent burden in Ewing sarcoma patients with clinically evident BM metastasis (N=6).</p>