Impact of Two Measures of Micrometastatic Disease on Clinical Outcomes in Patients with Newly Diagnosed Ewing Sarcoma: A Report from the Children's Oncology Group

Abstract

Purpose: 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.

Experimental design: 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 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.

Results: 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 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).

Conclusions: 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. Clin Cancer Res; 22(14); 3643-50. ©2016 AACR.

Figure 1

Dot plot of CD99+CD45− (bright+dim) percent in primary flow cytometry analytic cohort (N=109) compared to normal BM controls (N=22). Values were truncated if CD99+CD45− (bright+dim) percent >0.05 [truncated in N=6 (range of values = 0.071 – 1.1) from analytic cohort vs. N=0 from normal BM controls]; median values for each group is shown by horizontal crosses.

Figure 2

Kaplan-Meier estimated (A) event-free survival and (B) overall survival of flow cytometry cohort by CD99+CD45− (bright+dim) percent classified as “positive” or “negative” for BM micrometastatic disease according to upper limit from control subjects. Kaplan-Meier estimated (C) event-free survival and (D) overall survival of PCR cohort according to presence (“positive”) or absence (“negative”) of detectable EWSR1/FLI1 fusion transcript.

Figure 3

Box plots of IGF-1R (CD221) co-expression expressed as geometric mean fluorescence intensity (gMFI) on occult BM Ewing sarcoma cells (CD99+CD45− bright) vs. normal BM hematopoietic cells (CD45+).