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

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Presentation on theme: "Flow Cytometric Detection of Bone Marrow CD99+CD45- Cells in Ewing Sarcoma Steven DuBois, MD."— Presentation transcript:

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 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 CD CD

5 Flow Cytometry Detects A673 EWS Cells in Control PBMCs Count = 5 Count = 3 Count = 12 Count = 31 Count = 314 Count = 2975 CD45 CD99 ABC DEF 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) Median age (range)13 years (4 – 24 years) Male : Female12 : 5 Primary Site Extremity Chest wall Pelvis Other Metastatic Disease2 (both isolated lung)

9 CD99+CD45- Cells Detected in Bone Marrow from EWS Patients CD45 CD99 p = Median Percent Bone Marrow CD99+CD45- Cells (Range) Cases (n = 17) Controls (n = 6) 0.021% ( ) % ( )

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

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

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 Acknowledgements 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

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17 CD45 CD99


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