flow cytometric detection of bone marrow cd99+cd45- cells in ewing sarcoma
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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 - PowerPoint PPT PresentationTRANSCRIPT
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
~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
Disadvantages of PCR
Need to know type of EWS fusion transcript
Stability of mRNA
Can not further characterize the cells
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
100
101
102
103 4
CD45
100
101
102
103
104
CD
99
96.29
Flow Cytometry Detects A673 EWS Cells in Control PBMCs
Count = 5 Count = 3 Count = 12
Count = 31 Count = 314 Count = 2975
CD45
CD
99
A B C
D E F
DuBois et al, in press
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
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
Patient Characteristics (n = 17)
Median age (range) 13 years (4 – 24 years)
Male : Female 12 : 5
Primary Site
Extremity
Chest wall
Pelvis
Other
9
3
2
3
Metastatic Disease 2 (both isolated lung)
CD99+CD45- Cells Detected in Bone Marrow from EWS Patients
CD45C
D99
p = 0.0005
Median Percent Bone Marrow CD99+CD45- Cells (Range)
Cases
(n = 17)
Controls
(n = 6)
0.021% (0.0011-0.18)
0.00075% (0.00027-0.0014)
CD99+CD45- Cells Detected in Marrow from EWS Patients
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
Controls Cases
Percent CD99+CD45- Cells
CD99+CD45- Cells Detected in Marrow from EWS Patients
0
0.0025
0.005
0.0075
0.01
Controls Cases
Percent CD99+CD45- Cells
CD99+CD45- Cells Detected in Marrow from EWS Patients with Metastatic Disease
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
Controls Cases
Percent CD99+CD45- Cells
2 Patients with Isolated Lung Metastases Patient with Bone Marrow Metastasis
CD45C
D99
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
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
Acknowledgements
Co-InvestigatorsC. Lorrie EplingRobert GoldsbyChi BraunreiterDoug HawkinsHolcombe GrierKatherine MatthayElizabeth SinclairStephen Lessnick
MentorshipHolcombe GrierStephen LessnickKatherine Matthay
FundingSarcoma Foundation of
AmericaHope Street KidsCampini FoundationCOG / CureSearchUCSF CTSI KL2
CD45
CD
99