Excellent Prognosis in a Subset of Patients with Ewing Sarcoma

Author Manuscript Published OnlineFirst on April 5, 2011; DOI: 10.1158/1078-0432.CCR-10-3069 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Excellent prognosis in a subset of patients with Ewing sarcoma

identified at diagnosis by CD56 using flow cytometry

Ash Shifra1,4*, ,Luria Drorit1,2*, Cohen J Ian1,4, Goshen Yacov1,4, Toledano Helen1,4,

Issakov Josephine3,4, Yaniv Isaac1,2,4, Avigad Smadar1,2,4

1Department of Pediatric Hematology-Oncology, Schneider Children’s Medical Center of Israel, Petach Tikva, 2Molecular Oncology, Felsenstein Medical Research Center, Rabin Medical Center, Petach-Tikva 3 Department of Pathology, Tel Aviv Sourasky Medical Center, Tel Aviv, 4Sackler Faculty of medicine, Tel-Aviv university, Tel-Aviv, Israel

*: equally contributed to this work

Running title: CD56 as a prognostic marker in localized Ewing sarcoma

Address for correspondence: Shifra Ash MD, Department of Pediatric Hematology-

Oncology, Schneider Children’s Medical Center of Israel. 14 Kaplan Street, Petach

Tikva, Israel 49202. Phone 972 39253669 Fax 973 39253042 e-mail: n-

[email protected]

Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 5, 2011; DOI: 10.1158/1078-0432.CCR-10-3069 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Statement of Translational Relevance.

We have shown that multi-parametric flow cytometry, using CD99, CD90, and a

negative hematopoietic panel, provides a possible strategy for detecting circulating

Ewing sarcoma cells. There was a highly significant correlation between CD56

expression on these Ewing sarcoma cells in BM samples at diagnosis and progression

free survival for the whole group, for patients with localized disease and for patient

with localized non-pelvic disease.

This is the first report in Ewing sarcoma patients, reporting CD56 as a prognostic

marker for relapse in BM samples at diagnosis. CD56 expression in BM samples at

diagnosis could be used to identify ES patients predisposed to relapse, thus applying

treatment intensification and implementation of personalized therapy. Furthermore,

CD56 evaluation can identify a subgroup of patients with excellent prognosis, in

whom treatment reduction could carefully be considered.

Abstract

Purpose

Ewing sarcoma (ES) is considered a systemic disease with the majority of patients

harboring micrometastases at diagnosis. Multi-parameter flow cytometry (MPFC) was

used to detect ES cells in bone marrow (BM) of ES patients at diagnosis and to

evaluate the prognostic significance of CD56 expression in bone marrow samples.

Experimental Design

BM samples from 46 ES patients, 6 tumor aspirates, 2 ES cell lines and 10 control

BM samples were analyzed by MPFC. ES cells were identified by the combination of

CD45–/CD90+/CD99+. CD56 was evaluated on these cells using a cut off of 22%.

Results

BM samples obtained from all patients at diagnosis were found to be positive for

micrometastatic tumor cells assessed by CD99+/CD90+CD45– expression. 60% of

the BM samples harbored high CD56 expression. There was a highly significant

correlation between CD56 expression and progression free survival (PFS) (69% in

low/negative expression versus 30% in high expression groups, p=0.024). In patients

with localized non pelvic disease, those expressing low/negative CD56 had 100%

PFS versus 40% in the high expression group (p=0.02). By Cox regression analysis

CD56 was found to be an independent prognostic marker with an 11-fold increased

risk for relapse in patients with localized disease (p=0.006).

Conclusion

All samples contained cells that are positive for the CD99+/CD90+/CD45–

combination at diagnosis indicating that ES is a systemic disease. CD56 expression

could be used to reveal ES patients with excellent prognosis or patients predisposed to

relapse, thus improving treatment stratification and implementation of personalized

therapy.

Keywords: Ewing sarcoma, Multi-parameter flow cytometry, CD99, CD56,

Progression free survival

Introduction

Ewing sarcoma (ES) is the second most common primary bone tumor in children and

adolescents, belonging to the neuroectodermal tumor group known as ES family

tumors (EFT). Approximately 25% of patients have metastases at diagnosis, which is

a major adverse prognostic factor. Additional clinical adverse prognostic features

include pelvic primary site and poor response, defined as less than 90% necrosis, at

definitive surgery [1].

Despite aggressive multimodal therapy, one third of patients with a localized tumor

still may develop a recurrence or progress. Thus, ES should be considered a systemic

disease with the majority of patients harboring micrometastases already at diagnosis

[1, 2]. ES tumors are characterized by specific translocations that result in the fusion

of the EWS gene on chromosome 22q12 with different ETS-related genes, including

FLI-1, ERG, ETV1, E1AF and FEV [1, 3]. These chimeric transcripts have been

detected by reverse-transcriptase polymerase chain reaction (RT-PCR) for the

diagnosis of ES. Due to the high sensitivity of the RT-PCR assay, it can also be used

for detecting circulating tumor cells in peripheral blood (PBL) and bone marrow

(BM) samples [4-8]

Flow cytometry (FC) is an alternative sensitive method used routinely for the

detection of Minimal Residual Disease (MRD) in hematopoietic neoplasm [9, 10]. It

is also used for diagnosis and follow up evaluation in solid tumors including breast

and prostate cancer [11, 12] in adult patients and rhabdomyosarcoma and

neuroblastoma in pediatric patients [13-15].

The surface antigen CD99 (MIC2) is a marker of ES cells. CD99 is involved in cell

adhesion, migration, apoptosis and differentiation of T cells and thymocytes. CD99 is

also highly expressed on T-cell lymphoblastic leukemia/lymphoma and other

hematopoietic progenitors [16, 17] which, in contrast to ES cells, also express CD45,

the pan leukocyte common antigen. Thus the combination of both antigens CD99+

and CD45- can be used to evaluate ES tumor samples. Indeed, DuBouis et al.[18]

have recently demonstrated that ES tumor cells can be accurately detected in a very

low frequency of 1 in 100,000 normal cells in BM and PBL by FC using the

combination of CD99 positive and CD45 negative cells. CD90 or Thy-1 is a 25–37

kDa heavily N-glycosylated, glycophosphatidylinositol (GPI) anchored conserved cell

surface protein with a single V-like immunoglobulin domain, originally discovered as

a thymocyte antigen in mice. CD90 (a stem cell associated antigen) is expressed on

subsets of hematopoietic [19] and nonhematopoietic stem cells [20, 21] and was

found to be expressed in ES/PNET tumors and cell lines [22].

CD56, an isoform of neural adhesion molecule (NCAM), is an integral membrane

protein expressed by a variety of normal cell types, including a range of

neuroectodermal derivatives and natural killer cells. CD56 is expressed in a wide

spectrum of neoplasms including epithelial neoplasms, some cases of ES and

primitive neuroectodermal tumor (PNET) [23-26]. FC using CD99, CD56 and CD45

has been used to evaluate ES tumor samples by applying the profile CD99+/CD45 –

and CD99+/CD56+/CD45– [24, 27]. In addition, the coexpression of bright CD90

increases the accuracy of ES diagnosis [22].

In this study, we used Multi-parameter flow cytometry (MPFC) to evaluate the

presence of circulating ES cells in BM samples of ES patients at diagnosis using the

combination of CD99+/CD90+/CD45– and to assess the prognostic value of CD56

expression on these cells.

Patients and Methods

We retrospectively analyzed BM samples from 46 patients with ES, treated at SCMCI

from 1990 to 2008. In 6 of these patients, tumor aspirates were also available.

Mononuclear Cells (MNCs) extracted at diagnosis from BM of ES patients, tumor

aspirates and control BM samples were cryopreserved in DMSO in liquid nitrogen.

This study was approved by the local and national Ethics Committees.

The median age at diagnosis was 15.8 years (0.3-26). The primary site was extremity

in 19/46 (41.3%), pelvis-sacrum in 8/46 (17.4%), Askin tumor in 6/46 (13.1%), other

axial 10/46 (21.7%) and multifocal 3/46 (6.5%). Thirty five (76%) of patients

presented a localized disease (Table 1).

All patients were treated by an in house protocol which included 6 courses of

chemotherapy (Vincristine, Actinomycin-D, Cyclophosphamide, Doxorubicin

Ifosfamide and Etoposide), high radiation dose of 4500-6000cGy and surgery

Histopathological diagnosis was based on the appearance of small round cells;

immunohistochemical staining included CD99, Synaptophysin, Chromogranin, NSE.

All tumor samples were positive for EWS-FLI-1 transcript by molecular analysis.

Clinical staging was based on diagnostic imaging studies including x-rays, CT scans,

MRI and Tc99 bone scans. Median follow up was 60.9 months. Overall survival (OS)

and PFS for the whole group was 67.6 % and 50.5 % in 5 years, respectively.

Ten control BM samples were obtained from children that were evaluated for

hematological investigation and were found to be with no malignancy.

Sample preparation and staining

We implemented the MPFC methodology for MRD analysis [28] to evaluate ES cells

in patient`s BM obtained at diagnosis. All samples were coded and evaluated blindly.

Cells were stained as described previously, using five-color combination of eight

different antibodies: CD99-FITC (clone TU12; Becton Dickinson [BD] San Jose,

CA), CD90-PE (clone 5E10, BD), CD45 Per-CP (clone 2D1, BD), CD3,14,16,19-PE-

Cy7 (clones: UCHT1, RMO52, 3G8, J3-119 respectively, Beckman Coulter [BC],

Hialeah, FL), CD56-APC (clone NCAM 16.2 BD).

At least one million defrosted MNCs were stained and analyzed on a FACSCanto II

flow cytometer BD Biosciencesusing the Diva software for acquisition and analysis.

For sensitivity analysis serial dilutions of pre labeled and fixed ES cell line

(MHHES5) were performed in PBS. Desired amounts were spiked into control BM

which were then labeled with the five-color combination and fixed with CellFIX (BD)

[29].

MPFC analysis

MPFC analysis was performed using the gating strategy based on sequential removing

of non relevant cells as described in Figure 1. A debris exclusion gate was set on the

forward and side scatter (SSC) plot to define the total relevant MNCs. CD45–gate was

then drawn on a CD45 versus SSC plot to define the suspected ES cells. The CD45–

cells were gated again on a CD (3/14/16/19)-/dim versus SSC plot to exclude lineage

specific stem cells that are present in the BM. Cells defined as CD45–/CD3–/CD14–

/CD16–/CD19–were analyzed for the ES specific markers on a CD99 versus CD90

plot. We defined ES cells as CD45–/CD3–/CD14–/CD16–/CD19– and positive for

both CD99/CD90 (ES combination). ES positive cell were defined when a clear

population of more than 10 events was captured by the gating strategy [30]. The

proportion of ES cells was expressed as a percent of the total MNCs. A gate was

drawn on the double positive cells which were then analyzed for CD56 positivity on

CD56 versus CD99 and CD56 versus CD90 plots (Figure 1). The proportion of

CD56+ was expressed as a percent of the gated ES cells.

Cell lines

ES cell lines: SK-ES1 and MHHES5 were obtained from DSMZ (Leibuiz, Germany),

and cultured according to DSMZ recommendations.

Statistical analysis

ROC analysis was used for the determination of the cut-off values for the CD56

expression levels with relapse as the dependent variable and CD56 as the independent

variable. High or low expression levels were determined as higher as or lower than

the cut-off obtained. PFS by Kaplan Meier analysis was assessed by high versus low

CD56 expression levels. The correlations between CD56 expression and clinical

parameters as age, metastasis at diagnosis were evaluated by Fisher's exact test.

Multivariate analysis using Cox regression was performed for patients with localized

disease by the parameters age, primary site and CD56 expression. Kaplan Meier

analysis was used for PFS and OS of the whole cohort (PASW Statistics 18).

Results

Both cell lines were characterized as expressing CD99, CD90 and were negative for

the hematopoietic panel, consistent with our definition. All 6 tumor aspirates also

expressed both CD99 and CD90.

All control BM samples were negative for the CD99 and CD90 combination. By the

spiking experiments we reached the sensitivity of 1 in 100,000 normal cells (data not

shown).

ES micrometastatic cells in BM samples

46 BM samples obtained at diagnosis were evaluated for the ES combination and all

were found to be positive (≥0.001%), indicating that all BM samples harbor

micrometastatic ES tumor cells (range 0.001-0.4%)(Table 1).

No statistical differences could be shown in the OS or PFS of patients according to

the level of BM involvement, nor could any correlation with clinical parameters be

identified.

CD56 expression on ES cells

ES cell lines were also analyzed for CD56 which was found to be differentially

expressed as follows: SKES-100% and MHHES-43%.

CD56 was evaluated on ES cells in BM samples obtained at diagnosis from 45

patients. In one patient (# 25) the quality of the sample and low number of cells

impeded the evaluation of CD56. High or low expression was defined as higher or

lower than the cut-off of 22% that was defined by ROC analysis. Sixty percent of the

samples (27/45) harbored high CD56 expression. No significant correlation was found

between CD56 expression and clinical parameters as age, primary site and metastasis

at diagnosis (Table 1). A highly significant correlation between disease progression

and CD56 expression was detected by Kaplan Meier for the whole cohort. For the

group of patients harboring low/negative CD56 expression PFS at 10 years was 69%

versus 30% in the group with high expression (p=0.024)(Figure 2A). When analyzing

only the group with localized disease (n=35), those expressing low/negative CD56

had 81% 10 years PFS versus 38% in the high expression group (p=0.02) (Figure2B).

Furthermore, when excluding patients with pelvic disease out of the localized group

(n=30), the difference between the high to low expressing groups was even more

pronounced. 100% 10 years PFS for the low expression versus 40% for the high

expression (p=0.007) (Figure 3).

Cox regression analysis in the localized group determined that CD56 expression is an

independent prognostic marker with an 11-fold increased risk to relapse in patients

with high expression (Table 2).

Discussion

ES is an aggressive malignant bone tumor. A third of patients with localized disease

will progress. Currently, the clinical prognostic markers are inadequate to predict risk

of relapse. An improved tool is needed to define those patients that will eventually

relapse and offer them intensified treatment or to reduce treatment for patients with

good prognosis.

MPFC is a sensitive method for the quantitative detection of rare cell populations. It is

used to detect MRD and accordingly make clinical decisions in childhood acute

lymphoblastic leukemia [9,10]. This method has also been used to identify circulating

and/ or micrometastatic tumor cells in adult patients with carcinoma and was proved

to be a significant prognostic factor in patients with prostate cancer and breast

carcinoma [11, 12]. In pediatric patients, micrometastatic cells were detected in

neurobalstoma and rhabdomyosarcoma [13-15]. Only a few ES cases were reported

using FC for the diagnosis of ES tumors for the combination of CD45–/CD99+ [22,

27].

The feasibility of FC to detect circulating and micrometastatic ES cells was recently

published by DuBois et al [18]). They also showed that six color panel was more

sensitive than the two color assay in detecting residual ES cells with a background

rate of 0.00019%, just below 1 in 500,000 cells.

Our strategy was based on the negativity of CD45 and positivity of CD99 on ES cells.

In addition, we excluded hematopoietic stem cells and added another ES marker,

CD90. The accuracy of our method was achieved using a gated strategy of CD45-–

/CD3–/CD14–/CD16–/CD19– in addition to the positivity of CD90 and CD99 on ES

cells. Using this approach we could reach the same range of sensitivity of 1 in

100,000 as described by Dubois et al [18].

CD99 is expressed in the majority of cases of ES and is considered one of the best

diagnostic markers to date [31]. Interestingly, Rocchi et al. [32] have demonstrated

that CD99 is expressed on mesenchymal cells, which is considered the origin of ES

tumor. They have shown that CD99 is required for ES oncogenic phenotype by

preventing terminal neural differentiation. Chang et al. [22] have shown that CD90 is

highly expressed on ES cells, and has also been shown to be expressed on

mesenchymal stem cells [33]. We have detected CD99 and CD90 expression on all

cell lines and tumor aspirates studied.

All BM samples obtained from patients with localized and metastatic disease at

diagnosis were found to be positive for circulating ES tumor cells. There was no

correlation between the amount of ES cells in BM samples at diagnosis and survival.

In our previous study using RT-PCR, we could not show a correlation between the

presence of ES tumor cells in BM at diagnosis and outcome, but detected a highly

significant association between occult tumor cells in BM or PBL during follow up and

relapse [34] and also shown by de Alava et al. [35]. However, two reports from the

same group have identified a significant correlation between BM micrometastases in

localized patients and outcome [7, 36].

Although RT-PCR is the common technique for detecting circulating ES tumor cells,

there are significant advantages for MPFC including: rapid technique, no need to

identify a transcript, less labor-intensive comparing to RT-PCR, no need for the

extraction of RNA and can be detected in other body fluids.

CD56 is expressed in a variety of tumors. It has been detected in pediatric

malignancies like neuroblastoma, medulloblastoma, rhabdomyosarcoma, synovial

sarcoma, Wilm's tumor and hematopoietic malignancies [37-40]. Its expression in ES

is controversial. Using immunohistochemistry, ES tumors were found to be negative

for CD56 expression [40] and a few case reports of ES tumors reported positivity by

immunohistochemistry [41] or by FC [22, 24, 27]. This controversy could be

explained by the use of different monoclonal antibodies directed to different isoforms

on the NCAM molecule [38, 42].

We analyzed CD56 expression only on the defined ES cells by the combination

strategy. ES cell lines differentially expressed CD56 and 4 out of the 6 tumor

aspirates were positive for CD56. Twenty seven (60%) BM samples were CD56

positive, with a large range of expression: 3%-96%.

We detected a highly significant correlation between CD56 expression and disease

progression. Patients harboring low/negative expression had a significant better PFS.

Furthermore, when reanalyzing only the group with localized disease, those

expressing low/negative CD56 had even a better PFS at 10 years compared to patients

with high expression. When excluding the patients with the pelvic primary site, we

observed a more pronounced significant PFS: 100% 10 years PFS for the low

expressing group versus 40% for the high expressing group at 10 years (p=0.007).

Using Cox regression analysis by age, primary site and CD56 expression, high CD56

was found to be a significant independent prognostic marker for relapse in the

localized group.

To our knowledge this is the first study, in ES patients at diagnosis, reporting CD56

as a prognostic marker for relapse in BM samples. CD56 expression analysis can

identify prognostic groups and facilitate stratification of treatment. These results were

found using an aggressive treatment protocol of chemotherapy, surgery and

radiotherapy that have proved to be effective in delaying relapse in resected non

metastatic ES (unpublished data). It is important to analyze the significance of these

findings in patients treated on other treatment protocols.

Increased CD56 expression has been associated with a more aggressive form of solid

tumors, such as lung, ovarian and renal cell, and in hematopoietic malignancies -

lymphoblastic and myeloid leukemias [37, 43-45]. CD56 over expression in ES may

contribute to the aggressiveness of this childhood tumor.

It had been shown that high expression of NCAM (CD56) can be a target for therapy.

Indeed, there are several trials targeting the NCAM in malignancies, as small cell lung

cancer, neuroblastoma and glioma. The huN90-DMI immunotoxin is the most

advanced anti-NCAM therapeutic strategy and its current clinical development is

intensively promoted by pharmaceutical companies. There are also reports on naïve

humanized monoclonal antibodies such as huN901, and eight other monoclonal anti-

NCAM Abs using radioimmunotargeting with iodine. [37, 39, 43-45]

In conclusion, MPFC using CD99, CD90, and a negative hematopoietic panel

provides a possible alternative strategy for detecting micrometastatic Ewing sarcoma

tumor cells. CD56 expression in BM samples at diagnosis could be used to identify

ES patients predisposed to relapse, thus applying treatment intensification and

implementation of personalized therapy. Furthermore, CD56 evaluation can identify a

subgroup of patients with excellent prognosis, in whom treatment reduction could

carefully be considered.

Acknowledgement

This work was supported by the Margalit Josefsberg Foundation.

We thank Sara Dominitz for the English editing and Pnina Lilos for the statistical

analysis.

Legends to figures

FIGURE 1: MPFC strategy:

A. Debris exclusion in gate P1 on SSC/FSC dot plot.

B. Exclusion by CD45: CD45 negative cells are shown in P2 on CD45/SSC dot plot.

C. Exclusion by lineage specific stem cells out of CD45- cells: Selection of the CD3, CD14, CD16, CD19 negative cells by gate P3.

D. Cells fulfilling characteristics defined in gates P1, P2 and P3 are measured for CD99+CD90 positivity in gate P4 on CD99/CD90 dot plot.

E, F. Evaluation of CD56 on ES cells gated on CD56/99 and CD56/90 dot plots, shown in P5 and P6, respectively.

FIGURE 2: Kaplan Meier analysis for progression free survival by CD56 expression

A: The whole cohort of ES patients (n=45).

B: Localized ES patients (n=35).

High: above or equal to the cut-off of 22%; Low: under the cut-off of 22%

FIGURE 3: Kaplan Meier analysis for progression free survival by CD56 expression

in the non-pelvic localized ES patients (n=30). High: above or equal to the cut-off of

22%; Low: under the cut-off of 22%

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Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Downloaded from Fig 1 Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Author ManuscriptPublishedOnlineFirstonApril5,2011;DOI:10.1158/1078-0432.CCR-10-3069 clincancerres.aacrjournals.org on October 1,2021. ©2011 American Association forCancer Research. Downloaded from Fig 2 Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Author ManuscriptPublishedOnlineFirstonApril5,2011;DOI:10.1158/1078-0432.CCR-10-3069 clincancerres.aacrjournals.org A B

Low/negative expression 81%

High expression 38% High expression 38% bability bability o o o Hiihgh expressi on 30% o Pr Pr

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Fig 3 Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Author ManuscriptPublishedOnlineFirstonApril5,2011;DOI:10.1158/1078-0432.CCR-10-3069 clincancerres.aacrjournals.org

bability High expression 40% bb oo Pr Pro

p=0.007

Months Author Manuscript Published OnlineFirst on April 5, 2011; DOI: 10.1158/1078-0432.CCR-10-3069 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

TABLE 1: Clinical parameters and expression results of CD90/99 and CD56 No Age Primary Metas CD90/99 CD56 Relapse Follow (Years) site (%) (%) up 1 11.9 limb No 0.03 23 No 101 2 16.9 pelvis No 0.06 86 Yes 21 3 24 pelvis No 0.01 19 Yes 35 4 7.6 trunk No 0.003 56 Yes 33 5 1 limb No 0.007 12 No 50 6 23 pelvis No 0.006 12 No 89 7 2.3 limb No 0.004 0 No 86 8 16 limb No 0.008 69 No 78

9 19.7 pelvis No 0.008 3 Yes 62 10 5.8 trunk No 0.021 72 No 77 11 4.6 limb No 0.014 15 No 38 12 18.2 trunk No 0.08 97 No 95 13 14.4 limb No 0.004 30 Yes 99 14 15.9 limb No 0.058 36 No 88 15 23.8 trunk No 0.01 4 No 162 16 24 trunk No 0.007 4 No 33 17 9 limb No 0.06 76 Yes 28 18 16.8 trunk No 0.026 5 No 130 19 12.7 Limb No 0.004 0 No 158 20 15.9 Limb No 0.078 65 No 79 21 8.9 Limb No 0.068 43 Yes 21 22 7.4 Limb No 0.005 37 Yes 6 23 11.6 limb No 0.001 83 Yes 7 24 17.4 trunk No 0.005 74 No 242

25 15.3 pelvis No 0.001 No 238 26 19.5 limb No 0.01 67 Yes 31 27 14.3 trunk No 0.014 74 No 179 28 21.7 limb No 0.005 39 No 153 29 16.3 trunk No 0.08 16 No 242 30 24.3 limb No 0.15 21 No 168 31 3.7 chest No 0.005 96 Yes 39 32 25 chest lung 0.004 35 Yes 30 33 2.4 chest No 0.001 0 No 83 34 12 chest No 0.014 81 Yes 23 35 14 chest No 0.02 90 Yes 50 36 10.8 chest No 0.02 37 Yes 20 37 19.9 trunk lung 0.003 0 No 45 38 9.4 limb bone 0.022 0 Yes 8 39 20 Multifoc bone 0.003 14 Yes 15 al bone 40 13.6 limb lung 0.008 28 Yes 31 41 25.9 Multifoc bone 0.01 29 Yes 41 al bone 42 18.2 Multifoc bone 0.4 51 Yes 7 al bone 43 24.9 pelvis lung 0.016 13 No 39 44 9.6 limb lung 0.01 21 Yes 20 45 17.8 pelvis lung 0.005 36 Yes 22 46 21.7 pelvis bone 0.06 54 Yes 5

Age: at diagnosis; Metas: metastasis at diagnosis; CD56: expression on the CD90/99 positive cells only; Follow up: months from diagnosis untill relapse or untill last follow up.

TABLE 2: Univariate and Multivariate analyses of the Clinical Parameters and CD56 expression results for localized ES patients (n=35). Parameter Univariate Multivariate p p RR 95% CI CD56 0.02 0.006 11.8 2.0-68 Age 0.002 0.021 0.23 0.06-0.8 (years) Primary 0.08 0.002 17.3 2.9-102 site

P: p value, RR: relative risk to relapse; CI: confidence interval

Excellent prognosis in a subset of patients with Ewing sarcoma identified at diagnosis by CD56 using flow cytometry

Shifra Ash, Drorit Luria, Ian J. Cohen, et al.

Clin Cancer Res Published OnlineFirst April 5, 2011.

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