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PurposeFlow 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.Experimental designNewly 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 EWSR1/FLI1 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.ResultsThe 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 EWSR1/FLI1 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; P < 0.001).ConclusionsThe 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. Clin Cancer Res; 22(14); 3643-50. ©2016 AACR.

Abstract Background Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). Results Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4+cytokine+ cells and CD8+cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24%) for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82%) for samples with a mean of Conclusion ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

Background: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). Results: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4+cytokine+ cells and CD8+cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of >0.5% IFNγ+ T cells, and highest (57-82%) for samples with a mean of <0.1% IFNγ + cells. Conclusion: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays. [ABSTRACT FROM AUTHOR]