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Original Article

Bone marrow aspirations in Ewing

sarcomas: Are they still necessary?
A single‑center retrospective analysis and
review of the literature Bernadette
Breitegger,
Lukas A. Holzer,
ABSTRACT
Christine
Background and Objectives: Currently, one of the most useful prognostic indicators in Ewing sarcomas (ES) is the presence of Beham‑Schmid1,
metastatic disease at diagnosis. According to various clinical guidelines, the assessment of bone marrow (BM) metastases, using light Christian Urban2,
microscopy examination of bone marrow aspirates and biopsies (BMAB) is mandatory. However, the prognostic value of BM positivity Bernadette
is discussed controversially. Therefore, the primary aim of this study was to retrospectively review BM samples from patients with ES. Liegl‑Atzwanger1,
Materials and Methods: This retrospective single centre study included 31 patients that were newly diagnosed with ES between Andreas Leithner
2000 and 2014. Twenty-seven patients had skeletal ES and in 4 patients the tumour was localized in the soft tissue only. Metastases Departments of
at diagnosis were present in 5 out of 31 patients. BM samples were morphologically and immunohistochemically searched and Orthopaedic Surgery
screened for the presence or absence of BM metastases. Furthermore, in 15 of the 31 patients BM samples were still available and and 2Pathology,
were reanalysed, using nested-polymerase chain reaction. Medical University of
Graz, 1Department of
Results: All BM samples of our 31 ES patients, including the 5 metastatic patients, were, morphologically and immunohistochemically Pediatrics, Division of
tested negative for tumour cell appearance. The nested-PCR results were also negative in all of our 15 retested patients, including Pediatric Haematology
two patients with metastatic disease. and Oncology, Medical
University of Graz,
Conclusions: Based on our results and on the contradictory results reported in the literature we recommend a re-evaluation of the
Graz, Austria
necessity and the prognostic value of BMAB in the initial staging process of newly diagnosed ES patients.
For correspondence:
Dr. Bernadette
KEY WORDS: Bone marrow aspiration/biopsy, bone marrow metastases, Ewing sarcoma Breitegger,
Department of
Orthopaedic
Surgery, Medical
INTRODUCTION of <20% in patients with multiple bone metastases University of Graz,
predict a worse outcome than patients harboring Auenbruggerplatz 5,
Currently, one of the most useful adverse prognostic lung/pleura metastases which show 5‑year survival 8036 Graz, Austria.
indicators in Ewing sarcomas (ES) is the presence rates of 20%–40%.[4] It still remains unclear why E‑mail: bbreitegger@
hotmail.com
of metastatic disease at diagnosis.[1] At initial patients with isolated lung metastases fare better
diagnosis, between 20% and 25% of patients than patients with bone or BM metastases.[5]
show metastases, which occur in about 10% of
patients in the lung or pleura, and in another 10%, According to the 2012 ESMO clinical practice Submitted: 26-Aug-2016
bone/bone marrow (BM) metastases are found.[2] guidelines, taking BM aspiration/biopsy (BMAB) Accepted in Revised
BM metastases are defined by light microscopic is mandatory, although the prognostic value of Form: 24-Feb-2018
BM involvement in aspirate or biopsy samples.[3] positively tested samples has not yet been proven.[2] Published: 24-Oct-2018

By applying a multimodal treatment including

chemotherapy, surgery, and radiotherapy, the
prognosis of ES has improved over the past
decades. This led to survival rates of about
60%–70% in localized, but only about 20%–40%
in metastatic disease.[4] Five‑year survival rates
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Cite this article as: Breitegger B, Holzer LA, Beham-Schmid C, Urban C, Liegl-Atzwanger B, Leithner A. Bone marrow
aspirations in Ewing sarcomas: Are they still necessary? A single-center retrospective analysis and review of the literature. J
Can Res Ther 2020;16:713-7.

© 2018 Journal of Cancer Research and Therapeutics | Published by Wolters Kluwer - Medknow 713
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Breitegger, et al.: Bone marrow aspirations in Ewing sarcomas: Are they still necessary?

Corresponding to the EURO Ewing 99 study protocol from Table 1: Patients’ characteristics
2006, BMAB examination is also required in the staging Patient Sex Age at Localization of Metastases
process of ES. Furthermore, they define BM metastases number diagnosis primary tumor at diagnosis
by light microscopic evidence of BM involvement in any 1 Male 14 Pubis ‑
aspirate or trephine biopsy sample. Molecular evidence 2 Female 3 Metacarpal bone ‑
3 Male 8 Ilium ‑
(i.e., by real‑time polymerase chain reaction [RT‑PCR] analysis) 4 Male 23 Soft tissue (thigh) ‑
alone is, by definition of this protocol, not considered 5 Female 13 Tibia ‑
adequate for diagnosis of metastatic BM disease.[3] 6 Male 18 Femur ‑
7 Male 14 Ulna ‑
8 Male 19 Rib Bone
The primary aim of this study was to retrospectively review 9 Female 20 Rib ‑
BM samples from patients with ES and as second objective 10 Male 8 Calcaneus ‑
to review published literature concerning BM examination 11 Female 12 Tibia ‑
12 Male 1 Ilium Lung
in ES because the prognostic value of BM metastases in ES is 13 Female 25 Femur ‑
discussed controversially. 14 Male 11 Humerus ‑
15 Female 17 Fibula ‑
MATERIALS AND METHODS 16 Male 15 Humerus ‑
17 Male 14 Humerus ‑
18 Male 14 Clavicula ‑
Dataset 19 Female 22 Tibia ‑
Data were retrieved retrospectively by analysis of a dataset 20 Male 13 Femur ‑
including ES patients that were diagnosed and treated between 21 Male 10 Tibia ‑
22 Male 15 Soft tissue (lower leg) ‑
2000 and 2014. This dataset provided patient information 23 Male 15 Sacrum Lung
enclosing age, gender, and data about diagnosis and treatment 24 Male 15 Scapula ‑
of the tumor. The main attention was paid to the data 25 Female 10 Tibia ‑
26 Male 18 Ilium Lung+bone
concerning BMAB.
27 Female 18 Soft tissue (iliopsoas) ‑
28 Male 10 Rib ‑
A literature search was done using the Medline database 29 Male 10 Tibia ‑
to identify relevant literature, published from 1995 to July 30 Male 16 Sacrum Lung
31 Female 8 Soft tissue (upper leg) ‑
2015. The following terms were used: “Ewing sarcoma,”
“bone marrow,” “bone marrow metastases,” “bone marrow
aspiration/biopsy,” “staging,” and “predictive potential.” Furthermore, in 15 of the 31 patients, BM samples were still
Papers providing quantitative results were included, whereas available and were reanalyzed using nested PCR. Five of the
review articles were excluded and divided into groups, based 15 retested BM samples were available from the left and the
on their findings concerning the value of BM positivity in ES. right side.

Patients
The study included ES patients with molecular secured ES and
patients who underwent BMAB. The only exclusion criteria
were the lack of secured ES and BMAB evaluation. Based on
these criteria, the study population consisted of 31 newly
diagnosed ES patients. They were between 1 and 25 years
of age (median age, 13.83 years). Twenty‑one were male
and 10 were female. Twenty‑seven patients had skeletal ES,
and in 4 patients, the tumor was localized in the soft tissue.
Metastases at diagnosis were present in 5 patients. In 3 of
these 5 patients, the lungs were the only site of metastases.
In 1 of 5 patients, metastases occurred in the bone and 1 of
5 patients had a combination of lung and bone metastases.
Patients’ characteristics are shown in Table 1.
Bone marrow assessment
All BM samples were obtained at diagnosis, before
treatment. All samples were collected from the iliac crest
and were morphologically and immunohistochemically
examined by the pathologists. These retrospective findings
were searched and screened for the presence or absence of
BM metastases.
Ethics
The study was approved by the Ethics Committee.
RESULTS
Morphologically, there was no evidence of BM infiltration
by the known ES in any of the histopathological findings
of our 31 patients. Furthermore, the samples underwent
immunohistochemical studies, performed at the unfixed
material. In none of our 31 patients, immunohistochemistry
showed any evidence of BM infiltration by the known ES.
All 31 patients, also the 5 patients with metastases at
diagnosis, were morphologically and immunohistochemically
free of a tumor in the BM. PCR results were negative in
all of our 15 retested patients. These 15 patients included
two patients with metastatic disease (1 with bone and
1 with lung metastases). Results of morphological and
immunohistochemical analysis and PCR can be seen in Table 2.
By searching Medline database, 15 relevant studies were
identified and further analyzed [Table 3]. In 7 out of 15 studies,
no clear conclusion was drawn whether tumor positive tested

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Breitegger, et al.: Bone marrow aspirations in Ewing sarcomas: Are they still necessary?

Table 2: Results of immunohistochemistry and polymerase The ideal method to examine BM is not clear. Although
chain reaction analysis according to the 2012 ESMO clinical practice guidelines,
Patient Metastases at Morphological and PCR results BMAB taken at sites distant from the primary tumor or known
number diagnosis immunohistochemical metastatic lesions are mandatory, they state that the added
results
prognostic value of molecular positivity over light microscopic
1 Negative Negative
2 Negative Negative
evaluation has not yet been proven.[2]
3 Negative Negative
4 Negative Negative Wagner et al. reviewed other methods to assess BM involvement
5 Negative Negative in ES.[20] Additional to morphological and immunohistochemical
6 Negative Negative
7 Negative Negative
methods, BM evaluation by using PCR is a common molecular
8 Bone Negative Negative method.[20] Ten of 15 studies described in the literature research
9 Negative Negative right above also performed PCR, but corresponding to the EURO
Negative left Ewing 99 study protocol, molecular evidence (i.e., by RT‑PCR
10 Negative Negative right
Negative left analysis) alone is not considered adequate for diagnosis of
11 Negative Negative right metastatic BM disease.[3] As described by DuBois et al., a new
Negative left method to detect ES cells in BM is flow cytometry, where BM
12 Lung Negative ‑ cells are stained for cluster of differentiation antigen (CD)
13 Negative Negative
14 Negative ‑ 99 and CD45 to detect CD99+  CD45−  cells.[21] Ash et  al.
15 Negative ‑ reported the use of multiparameter flow cytometry to detect ES
16 Negative ‑ cells in BM. This method identifies CD99+ and CD90+ tumor
17 Negative ‑
cells and being negative for CD45 and other hematopoietic
18 Negative ‑
19 Negative ‑ markers.[16] This study, performed in 2001, is the only one where
20 Negative ‑ all BM samples (46/46) were tested positive for micrometastatic
21 Negative ‑ tumor cells.[16] Furthermore, in this study, 27/45 (60%) BM
22 Negative ‑
23 Lung Negative ‑
samples were found to show high CD56 expression, and
24 Negative ‑ a significant correlation between disease progression and
25 Negative ‑ CD56 expression was described.[16] Another potential method
26 Lung and bone Negative ‑ to asses BM in ES patients may be the use of FISH, which
27 Negative Negative right
Negative left
identifies translocations involving the EWS gene, but there
28 Negative Negative are no current studies available with regard to this method.[20]
29 Negative ‑ A noninvasive method to assess bone and/or BM involvement
30 Lung Negative Negative right is F18‑fluorodeoxy‑D‑glucose (FDG)‑PET, as demonstrated in the
Negative left
31 Negative ‑
study by Newman et al., where 0/57 nonmetastatic patients
PCR=Polymerase chain reaction showed BM involvement by FDG‑PET.[1]

BM has any influence on the prognosis in ES patients.[6‑12] Five of
15 studies correlated BM positivity with worse outcome, which
was shown in 2 studies only in metastatic patients, in 1 study
only in nonmetastatic patients, and in 2 of these 5 studies in
both metastatic and nonmetastatic patients.[13‑17] Three out of
15 studies did not show a negative correlation over positive
tested BM and disease progression, and 2 of these even suggest
the elimination of BMAB in nonmetastatic ES patients.[1,18,19]
DISCUSSION
Corresponding to international clinical guidelines, BMAB
examination is mandatory in the staging process of ES.
In our study, we retrospectively analyzed the presence of tumor
cells in BM samples from patients with ES.
As the prognostic value of BMAB in ES patients is discussed
controversially, we tried to better specify the need of this
investigation in the staging process.
In our study, 0/31 ES patients (5/31 with metastases) showed BM
involvement using morphological and immunohistochemical
methods, and also, the nested‑PCR results were negative in all
of our 15 retested patients (2/15 with metastases) at the time
of diagnosis. These results are different to the 10/15 studies
that also used PCR methods to examine BM because in all of
them, positive BM samples were found.[6,8‑13,15,17,18]
Limitations of our study and the 10 studies using PCR are
the small cohorts (including 14–131 ES patients) and the
retrospective study design. Neither the number of patients in
our study nor the used method seems to be the reason that
no positive BM sample was found in our patients.
Altogether the 15 studies included 731 (100%) ES patients
and BM involvement was found in 209/731 (28.59%) patients.
127/209 (60.76%) BM‑positive patients had known metastases,
whereas BM positivity was seen in only 82/209 (39.23%)
patients without distant metastases. According to these
results, BM involvement seems to be more frequent in
metastatic patients. Fagnou et al. also concluded that RT‑PCR

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Breitegger, et al.: Bone marrow aspirations in Ewing sarcomas: Are they still necessary?

Table 3: Results of 15 studies concerning BM examination in ES

Study Testing Method No. of all No. of BM No. of BM No. of BM + in No. of BM + in
Patients + Patients ‑ Patients metastatic Patients non‑metastatic Patients
Pfeiderer et al. 1995 RT‑PCR 16 6/16 10/16 5/6 1/6
Oberlin et al. 1995 Morphological 59 13/59 46/59 12/13 1/13
Peter et.al 1995 Nested‑PCR 15 4/15 11/15 4/4 0/4
Zoubek et al. 1995 RT‑PCR 14 6/14 8/14 4/6 2/6
West et al. 1997 RT‑PCR 22 5/22 17/22 2/5 3/5
Sumerauer et al. 2001 RT‑ PCR 22 8/22 14/22 5/8 3/8
Athale et al. 2001 RT‑PCR 26 7/26 19/26 7/7 0/7
Fagnou et al. 1998 RT‑PCR 43 14/43 29/43 8/14 6/14
Oberlin et al. 2006 ‑ 97 23/97 74/97 23/23 ‑
Avigad et al. 2003 RT‑PCR 14 6/14 8/14 ‑ 6/6
Ash et al. 2001 MPFC 46 46/46 0/46 11/46 35/46
Schleiermacher et al. 2003 RT‑PCR 131 36/131 95/131 18/36 18/36
Zoubek et al. 1998 RT‑PCR 35 16/35 19/35 9/16 7/16
Newman et al. 2013 ‑ 75 6/75 69/75 6/6 0/6
Kopp et al. 2014 ‑ 116 13/116 103/116 13/13 0/13
Results 9/15 RT‑PCR 731 209 522 127 82
1/15 Nested‑PCR 100% 28.59% 71.40% 60.76% 39.23%
1/15 MPFC
1/15 Morphological
3/15 Unknown

positivity is frequently found in metastatic ES patients and
suggested that the monitoring of BM at diagnosis might be
an important criterion for the staging.[13]
Although BMAB generally is a simple and safe procedure,
there is a small risk of complications, and the intervention
can have considerable impact on individual patients.[22] Other
remarkable factors in proceeding BMABs is the need of time,
stuff, and equipment.
to the conclusion to stop carrying out BMAB in nonmetastatic
ES patients.[24]
In our retrospective study, all BM samples of our 31 ES patients,
including the 5 metastatic patients, were morphologically
and immunohistochemically tested negative for tumor cell
appearance. The nested‑PCR results were also negative in all
of our 15 retested patients. These 15 patients included two
patients with metastatic disease.

As far as a negative prognostic effect of positive tested BM
in ES is concerned, studies provide controversial results.
Only 3/15 studies, identified by the literature search, found
a negative effect of positive BM results.[15‑17] Avigad et  al.
showed that positive RT‑PCR BM results were correlated with
disease progression and recommended the serial monitoring
with RT‑PCR for the prediction of disease recurrence.[15]
Schleiermacher et al. also recommended the search for occult
tumor cells in the staging of ES patients.[17]
Ours, and other studies, concerning this topic, are limited by
the small cohort and by the retrospective study design.
CONCLUSION
Based on our results and on the contradictory results reported
in the literature, we recommend a reevaluation of the necessity
and the prognostic value of BMAB in the initial staging process
of newly diagnosed ES patients.

In contrary to these recommendations, Zoubek et al. excluded
a correlation of RT‑PCR BM positivity and early relapse in ES
patients.[18] Moreover, in the study by Kopp et al., 0/85 patients
without any imaging evidence of osseous metastases had BM
involvement.[19] These data indicate futility, not utility of BM
metastases examination, and the authors suggest that BMAB
may not be further required in the initial staging process of
ES patients considered nonmetastatic by imaging.[19] Referring
to this study, Peter Anderson published an article evaluating
the findings of BM examination in ES. Anderson therefore
recommends to first do modern imaging studies such as
chest CT and FDG‑PET, and if no metastases are found by
these imaging modalities, BM analyses can be considered
unnecessary.[23] In those patients with metastatic disease,
the utility of BM analyses is uncertain.[23] Although it is still
recommended in ongoing clinical trials, Valvi and Ziegler came
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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