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