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Flow Cytometric Detection of Bone Marrow CD99+CD45- Cells in Ewing Sarcoma

Flow Cytometric Detection of Bone Marrow CD99+CD45- Cells in Ewing Sarcoma. Steven DuBois, MD. PCR Detection of EWS Cells. Ewing sarcoma cells detectable in blood and bone marrow by PCR for EWS fusion transcripts

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Flow Cytometric Detection of Bone Marrow CD99+CD45- Cells in Ewing Sarcoma

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  1. Flow Cytometric Detection of Bone Marrow CD99+CD45- Cells in Ewing Sarcoma Steven DuBois, MD

  2. PCR Detection of EWS Cells • Ewing sarcoma cells detectable in blood and bone marrow by PCR for EWS fusion transcripts • ~30% of patients with non-metastatic Ewing sarcoma have detectable EWS fusion transcripts in bone marrow • Presence of EWS fusion transcript in bone marrow may be prognostic

  3. Disadvantages of PCR • Need to know type of EWS fusion transcript • Stability of mRNA • Can not further characterize the cells

  4. 4 10 96.29 3 10 2 10 CD99 1 10 0 10 0 1 2 3 4 10 10 10 10 CD45 EWS Cells are CD99+CD45- • Nearly universal expression of CD99 on Ewing sarcoma cells • Absence of CD45 • CD99+CD45- phenotype used to characterize Ewing primary tumors by flow cytometry

  5. Count = 12 Count = 3 Count = 5 A B C Count = 2975 Count = 314 Count = 31 D E F CD99 CD45 Flow Cytometry Detects A673 EWS Cells in Control PBMCs DuBois et al, in press

  6. Objective • To quantify the percentage of bone marrow CD99+CD45- cells in patients with newly diagnosed Ewing sarcoma without clinically evident bone marrow metastatic disease

  7. Methods • Mononuclear cells isolated from fresh marrow • Patients with new Ewing sarcoma • Controls with other malignancies undergoing bone marrow aspiration • Standard preparation for flow cytometry • Stained with commercially available flow cytometry antibodies for CD99 and CD45 • Collect 2-9 million events per sample • CD99+CD45- gate based on positive control from A673 cell line

  8. Patient Characteristics (n = 17)

  9. CD99+CD45- Cells Detected in Bone Marrow from EWS Patients CD99 CD45 p = 0.0005

  10. CD99+CD45- Cells Detected in Marrow from EWS Patients

  11. CD99+CD45- Cells Detected in Marrow from EWS Patients

  12. CD99+CD45- Cells Detected in Marrow from EWS Patients with Metastatic Disease CD99 CD45 2 Patients with Isolated Lung Metastases Patient with Bone Marrow Metastasis

  13. Conclusions • Patients with newly diagnosed Ewing sarcoma appear to have increased numbers of marrow CD99+CD45- cells • Available evidence suggests that these CD99+CD45- cells are Ewing sarcoma cells

  14. Next Steps • Comparison with RT-PCR ongoing • Evaluation of controls without malignancy ongoing • Cell sorting and evaluation by FISH for EWSR1 translocations ongoing • Evaluation of prognostic impact planned

  15. Co-Investigators C. Lorrie Epling Robert Goldsby Chi Braunreiter Doug Hawkins Holcombe Grier Katherine Matthay Elizabeth Sinclair Stephen Lessnick Mentorship Holcombe Grier Stephen Lessnick Katherine Matthay Funding Sarcoma Foundation of America Hope Street Kids Campini Foundation COG / CureSearch UCSF CTSI KL2 Acknowledgements

  16. CD99 CD45

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