Hindawi Publishing Corporation
Volume 2012, Article ID 780129, 8 pages
Assessmentof MinimalResidualDisease inEwingSarcoma
LarsM. Wagner,1TeresaA.Smolarek,2Janos Sumegi,3andDanielMarmer3
1Division of Pediatric Oncology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine,
3333 Burnet Avenue, Cincinnati, OH 45229, USA
2Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine,
3333 Burnet Avenue, Cincinnati, OH 45229, USA
3Division of Bone Marrow Transplantation, Cincinnati Children’s Hospital Medical Center,
University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
Correspondence should be addressed to Lars M. Wagner, firstname.lastname@example.org
Received 15 August 2011; Accepted 27 October 2011
Academic Editor: R. Pollock
Copyright © 2012 Lars M. Wagner et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Advances in molecular pathology now allow for identification of rare tumor cells in cancer patients. Identification of this minimal
residual disease is particularly relevant for Ewing sarcoma, given the potential for recurrence even after complete remission is
achieved. Using RT-PCR to detect specific tumor-associated fusion transcripts, otherwise occult tumor cells are found in blood or
bone marrow in 20–30% of Ewing sarcoma patients, and their presence is associated with inferior outcomes. Although RT-PCR
has excellent sensitivity and specificity for identifying tumor cells, technical challenges may limit its widespread applicability. The
use of flow cytometry to identify tumor-specific antigens is a recently described method that may circumvent these difficulties. In
this manuscript, we compare the advantages and drawbacks of these approaches, present data on a third method using fluorescent
in situ hybridization, and discuss issues affecting the further development of these strategies.
in children and young adults. The majority of ES patients
and pathologic examination of the bone marrow. However,
treatment with aggressive surgical removal alone cures only
undetected tumor cells in tissues such as lungs or bone
marrow that were present at least transiently in the blood.
Chemotherapy is used to eradicate this minimal residual dis-
ease (MRD), although clinicians have no routine method for
knowing the extent of MRD which remains in any given
patient. If a reliable, sensitive, and widely applicable assay
could be developed for MRD detection in ES, there would be
several obvious applications. First, since the finding of MRD
in patients with localized tumors is associated with worse
outcome , these patients could be identified early on for
more appropriate high-risk therapies. Second, MRD testing
could be used to assess patients for ongoing response to
chemotherapy , particularly after surgical removal of tu-
in tumor characteristics. Third, identification of the return
of low levels of disease may allow for early identification of
relapsing patients who have already completed therapy [4–
6]. Finally, MRD assessment may be beneficial with clinical
decision making in patients with equivocal imaging findings,
such as nonspecific lung nodules identified on computed
tomography scans , as it may support the diagnosis of
Therefore, given all these potential benefits, investigators
have tried for the past two decades to identify methods that
are not just sensitive and specific, but widely applicable and
feasible in multicenter trials. Ewing sarcoma is well suited
for such investigation, given its characteristic genetic and
immunophenotypic features which allow for distinction of
Table 1: Summary of Key Studies Using RT-PCR for MRD Detection in Ewing Sarcoma.
31% had MRD in either blood or marrow at diagnosis at diagnosis
38% had MRD in BM, while only 6% had CTC at diagnosis
25% of newly diagnosed localized pts had MRD in blood or marrow, compared to 50% with
26% of patients had MRD in blood at diagnosis, but this was not correlated with clinical features or
outcome.33% of patients had BM MRD, with worse outcome
MRD in blood and/or marrow developed prior to clinical progression
7/23 (30%) had BM MRD at diagnosis, but this did not predict relapse
Survival was correlated with speed of clearance of MRD in blood and BM
MRD in marrow found in 31% of localized and 50% of metastatic pts
Quantitative RT-PCR can be used to measure efficacy of stem cell purging methods
MRD in blood or BM at diagnosis is associated with worse survival in patients with otherwise
43% of pts had marrow MRD at diagnosisMRD developed prior to clinical recurrence in 10 of 11
Tumor cells frequently contaminate stem cell harvests, and are associated with relapse after
transplantation.Relapse is preceded by MRD in BM and/or blood
West  28
Fagnou  67
De Alava 
MRD, minimal residual disease; BM, bone marrow.
tumor cells from normal hematopoietic cells. In this manu-
common methodologies employed for MRD detection in
ES. In addition, we present preliminary data using a third
molecular assay and describe an ongoing clinical trial de-
signed to directly compare these assessment strategies.
2.RT-PCRfor MRD Detection
The majority of studies to assess MRD in ES patients have
focused on the use of reverse transcriptase-polymerase chain
reaction (RT-PCR) to identify tumor-specific fusion tran-
scripts. This method is based on the fact that approximately
85% of ES tumors are characterized by the EWS-FLI1
usually have other partners for EWS, including ERG, ETV1,
E1AF, and FEV. RT-PCR is attractive for use in MRD detec-
determined in spiking experiments to be one tumor cell in
one million mononuclear blood cells . In the largest
RT-PCR study to date, 20% of 107 ES patients who were
considered to have localized tumors using conventional as-
sessments did indeed have evidence of micrometastatic dis-
ease in the peripheral blood . Interestingly, 19% of such
patients also had MRD identified in the bone marrow,
although there was incomplete overlap between those with
MRD at either site. Importantly, patients with MRD at either
site had worse event-free survival compared to other patients
with localized disease, thus showing the potential utility of
MRD assessment as a prognostic indicator in a prospective
study. Multiple other smaller RT-PCR trials have confirmed
that up to one-fourth of newly diagnosed patients with ap-
parently localized tumors have MRD detectable by RT-PCR
in blood or marrow [2, 5, 9–14]. Other important applica-
tions demonstrated with RT-PCR testing include the ability
to assess the efficacy of induction chemotherapy regimens
 as well as novel purging techniques for peripheral blood
strated that MRD testing can identify relapse in patients
before it is clinically apparent by conventional imaging stud-
ies [5, 6, 16]. Table 1 summarizes some of the important RT-
PCR studies done to date, and these trials provide confir-
mation of the potential clinical relevance of MRD testing in
Limitations of RT-PCR include the potential for contam-
ination causing false positive results as well as degradation
of mRNA resulting in false negative results . The latter
may be particularly important for multicenter trials in which
same-day testing is not available. Another potential draw-
back of RT-PCR is that prior knowledge of the patient’s spe-
cific translocation is needed so that the appropriate primer
sets can be used (EWS-FLI1 versus EWS-ERG versus other).
Without this knowledge, interpretation of negative test
results is difficult. Historically, RT-PCR had often been per-
formed on initial tumor biopsies as a confirmatory test to
support the diagnosis of ES. However, because of technical
limitations such as sample size, tissue viability, or absence of
frozen tissue, RT-PCR of biopsy material is not always feasi-
ble. For example, in the largest multicenter study of RT-
PCR to detect MRD in ES, only 117 (68%) of 172 patients
had adequate tissue allowing identification of translocations
by RT-PCR . It is likely that this number may continue
to decrease given the now widespread use of fluorescent in
situ hybridization (FISH) probes to identify translocations
involving EWS , which can readily be done on paraffin-
One way to circumvent the requirement for knowledge
of the specific translocation partners is to instead assess for
individual genes universally expressed in tumor cells but not
hematopoietic cells. Cheung et al. used gene expression array
Figure 1: Sequential gating to identify Ewing sarcoma cells. Cultured A673 cells undergo sequential gating to identify Ewing sarcoma cells.
Mononuclear cells are separated from blood or marrow by density gradient centrifugation, stained with monoclonal antibodies (CD99 PE,
CD45 FITC, CD14 PerCP, CD34 APC), exposed to anti-PE magnetic microbeads to enrich CD99+ cells using MACS technology (Miltenyi
the CD99 bright positive CD45 negative tumor cells as shown in this example of A673 Ewing sarcoma culture cells.
data to identify 3 such genes meeting these criteria: STEAP1,
CCND1, and NKX2-2 . The expression of at least one
of these 3 genes in histologically negative bone marrow
samples from 35 Ewing sarcoma patients was associated with
progression-free and overall survival. Additional follow-up
studies using this approach have not yet been reported.
3.Flow Cytometry for MRD Detection
Another strategy that obviates the need to know the specific
translocation is to use multiparameter flow cytometry to
identify surface expression of tumor cell antigens. For exam-
ple, CD99 is universally present on ES tumor cells, and im-
munostaining for this protein has routinely been used to
confirm the diagnosis of ES in primary tumor samples .
However, since CD99 is also expressed on some blood cells
as well, negative selection for the leukocyte common antigen
CD45 is used to exclude hematopoietic cells. Further, to
reduce the low level background positivity seen in normal
blood and marrow samples, an additional sequential gating
strategy is used with a viability dye to remove dead cells,
CD14 to exclude monocytes, and CD34 to remove early
hematopoietic progenitors which may not yet express CD45.
This strategy was used in the first published report of flow
cytometry for MRD detection in ES by Dubois et al. .
They showed that residual ES cells from two different cell
lines can reliably be detected in spiking experiments of pe-
ripheral blood and bone marrow at the level of 1 tumor
cell in 500,000 or 1 tumor cell in 10,000 mononuclear cells,
We have instituted a clinical trial which uses the se-
quential gating strategy employed by Dubois and shown in
Figure 1. In addition, we have modified the assay by in-
corporating magnetic microbeads to enrich the tumor cell
concentration in the residual sample. Variations on this en-
richment approach have been described previously  and
can increase the confidence at which low numbers of tumor
cells can be identified. Figure 2 demonstrates how identifica-
tion can be improved through enrichment of CD99+ cells.
Notably, when enrichment techniques are used, sensitivity in
spiking experiments is similar or better to that achieved with
RT-PCR, with identification of tumor cells at the range of 1
in one million or more blood mononuclear cells.
Figure 2: Example of how enrichment can improve the identification of cultures A673 Ewing sarcoma cells mixed with peripheral blood
mononuclear cells: (a) No tumor cells are identified in a healthy volunteer blood sample analysis not containing tumor cells (negative
one A673 Ewing sarcoma cell per 1 × 106pbmc. (c) In contrast, use of enrichment technique allows for confident identification of tumor
cells (e.g., cluster of 5 or more events) that are CD99+/CD45- in a sample containing one A673 cell per 1 × 106pbmc. (d) Positive control
containing only A673 tumor cells.
Ash and colleagues have recently reported an alternative
flow cytometry method which identifies tumor cells express-
ing both CD99 and CD90 but which are negative for a
hematopoietic panel including CD45, CD3, CD14, CD16,
and CD19. CD90 is a cell surface protein expressed on some
hematopoietic and nonhematopoietic stem cells as well as
Ewing sarcoma cells . They assessed previously frozen
archival bone marrow samples from 46 patients, including
35 with localized tumors, as well as 10 control samples
remained negative, CD99+/CD90+ cells were identified in
all tested cell lines and patient samples. The range of tumor
burden identified in the patient samples was 0.001–0.4%,
and the reported sensitivity of the assay using spiking exper-
iments was 0.001% (one tumor cell in 100,000 mononuclear
cells). Tumor cells identified by this method were then tested
for expression of CD56, which is an isoform of neural cell
adhesion molecule (NCAM) found in natural killer cells and
neuroectodermal derivatives, including Ewing sarcoma .
Sixty percent of the 45 diagnostic samples had high levels of
and this identified a group with greater risk of recurrence. In
fact, in this study high CD56 expression in CD99+/CD90+
cells was determined to be an independent prognostic
marker withan 11-fold risk of relapse.Although theseresults
should be confirmed in additional studies, they underscore
that identification of molecular prognostic markers may be
another potential application of flow cytometry.
The fact that prior knowledge of a patient’s specific
a relevant MRD assessment tool for all ES patients. In addi-
tion, the assay is rapid and less labor-intensive than RT-PCR,
uses commercially available antibodies, and is well suited
for overnight delivery and analysis at a central laboratory.
For example, in children with acute lymphoblastic leukemia,
flow cytometry performed in a central reference laboratory
to assess response to induction therapy has been a feasible
and reliable prognostic marker in multi-institutional studies
 and has become part of the risk assessment strategy on
Children’s Oncology Group trials.
4.FISHfor MRD Detection
Another potential method toassessMRDis theuseofa FISH
break-apart probe to identify translocations involving the
Figure 3: Use of FISH to detect Ewing sarcoma cells. (a) Fluorescence in situ hybridization (FISH) signal pattern for normal cells using the
EWSR1 break-apart probe (Abbott Molecular) showing two fusion signals (red and green signal next to each other with little to no gap in
between the signals), which is the normal pattern. (b) FISH signal pattern from normal cells with an occasional false-positive signal pattern
(separation of one of the red and green signal pairs with a gap between the two signals wider than the size of one signal alone; see arrows)
for EWSR1 rearrangement. (c) In a sample containing Ewing sarcoma, there is widespread separation of one signal pair in multiple tumor
cells (labeled as 1R1G1F), compared to normal cells (labeled as 2F).
to pathological diagnosis of ES in primary tumor samples.
for EWS, recent studies suggest the specific translocation
partner does not hold prognostic significance for patients
treated with contemporary therapy , and so knowledge of
which gene fuses with EWS may no longer be relevant for
the routine care of ES patients. FISH can also be readily per-
formed on peripheral blood or bone marrow samples and
has been used to monitor MRD in leukemia patients .
However, there are no previous reports to our knowledge of
using FISH in ES for this purpose.
In our institution, up to 500 cells are routinely counted
when testing for minimal residual disease, which by defini-
tion limits the sensitivity to this number. However, potential
advantages of FISH testing include the ability to easily test
archived samples and the clear visual conformation of the
characteristic tumor-specific change in the EWS gene. How-
ever, even this can sometimes be difficult, depending on the
probe being used. A false positive interpretation may occur
due to DNA decondensation, which may cause the probes to
be sufficiently separated to mimic a true break-apart event.
neticist and must be carefully considered when interpreting
due to this stretching artifact.
At our institution, we have conducted FISH testing on
bone marrow aspirate samples from a limited number of ES
patients for the past 5 years, using the Vysis EWSR1 dual-
(Abbott Molecular, Abbott Park, IL). The tests were obtained
for clinical reasons at the discretion of the treating physician
and so were not ordered in any systematic fashion. In fact,
testing was not necessarily done on consecutive patients, or
even on all samples from an individual patient. Generally,
bone marrow samples were pooled together from both sides
for a single analysis. FISH testing was performed on 21 bone
marrow aspirates from 9 ES patients with either newly diag-
nosed or relapsed disease who were undergoing evaluations
for routine clinical care at Cincinnati Children’s Hospital.
Of these 21 pooled samples, 14 were negative for tumor by
both standard pathology assessment and FISH. In 6 samples
from 3 patients, likely tumor cells were identified by FISH
alone, with no tumor identified on conventional pathology
evaluation. In those patients, the percentage of cells reported
with possible EWSR1 rearrangement ranged from 0.2% to
patient sample had unequivocal tumor cells identified by
morphology on the bone marrow aspirate and biopsy but
was negative by FISH. The reason for this false negative re-
mains unclear, as FISH readily showed the characteristic
EWSR1 break apart in the primary bone tumor, as well in
a subsequent bone marrow sample done after induction
chemotherapy, in which a low level of residual tumor cells
was identified despite conventional morphology showing
bone marrow remission. We conclude from this limited pre-
liminary data that FISH analysis may detect tumor at low
levels not appreciated by conventional morphology in 29%
of samples, although one false negative test did occur.
Because the ideal method of MRD assessment in ES is
unknown at this time, we are currently performing a trial
which prospectively compares RT-PCR versus flow cytome-
try versus FISH in blood and marrow samples collected from
ES patients. Results will be compared between methods as
well as with bone marrow pathology reports and imaging
studies to correlate the utility of MRD testing with other
standard methods of disease assessment. Multiple institu-
tions are participating, which will allow us to assess the feasi-
day in a central laboratory.
There are several issues which must be worked out for MRD
assessment to have broad utility in ES. First, it is unclear
which site (blood or bone marrow) will ultimately provide
the greatest clinical relevance. In patients with extensive
tumor burden, assessment of either site is likely to yield the
same result, although these patients will benefit the least
from MRD testing because their disease is already clinically
apparent. For patients diagnosed with initially localized dis-
ease, the impact of minimal bone marrow involvement on
outcome has been inconsistent in smaller studies [5, 14].
However, results were more convincing in the largest trial
to date , which reported a decrease in 2-year disease-free
survival from 80% versus 53% when bone marrow MRD
testing was positive (P = 0.043). It is possible that this may
reflect that the impact on outcome is only apparent when
a sufficiently large number of patients are tested. Another
factor potentially leading to variable results is that bone mar-
row involvement in ES is more heterogeneous than that in
leukemia, and it is common for morphology assessments of
disease to differ between sides, and between the aspirates
and core biopsies. This was evident in our institutional
experience using FISH, in which one patient had aspirates
There is somewhat less data available regarding analysis
of circulating tumor cells in ES. As with bone marrow a con-
vincing effect on survival being related to circulating tumor
cells at diagnosis is seen in larger  but not some smaller
studies [5, 6]. Collection of blood samples is far less cumber-
csome for patients than bone marrow, and is well suited
for long-term monitoring either during or after completion
of therapy. In fact, the latter approach may be particularly
relevant, as several patients have been reported to have circu-
lating tumor cells prior to clinically apparent relapse [5, 6,
16]. In one of the larger studies, 10 of 11 patients with recur-
rence had tumor cells identified in blood or bone marrow
by RT-PCR prior to overt relapse, with a median time lag of
4.5 months (range 1–24 months) . In our current trial,
we are performing peripheral blood MRD evaluations any
time patients undergo imaging assessments (at diagnosis,
on therapy, or after therapy), while bone marrow testing is
only performed when marrow samples would be routinely
obtained for clinical purposes.
Quantification of RT-PCR results has not been generally
reported, with the exception of Merino et al., who used real-
time quantitative RT-PCR to estimate the effectiveness of a
bone marrow purging method . It is possible that this
approach would provide standardization of methodology
and consistency in determining exactly what constitutes a
positive test result. Similar standardization attempts would
be helpful for flow cytometry, given the difficulties in inter-
preting results when there are only one or two events in the
Another question is whether cells identified by these
methods are truly cancer cells, as each assay has the potential
EWS changes not found in hematopoietic cells, contamina-
tion during RNA collection and testing may occur. For FISH,
changes in the EWS gene during decondensation of DNA can
cause an occasional cell to appear as if there may be a true
rearrangement, as discussed earlier and noted in Figure 2.
For flow cytometry, despite the use of a panel of markers to
exclude hematopoietic cells, there is always the possibility of
illegitimate transcription of these hematopoietic markers in
tumor cells. In fact, in the most recent report by Ash et al.
, flow cytometry was reported to identify tumor cells in
all 35 diagnostic bone marrow samples from patients with
localized disease, and this incidence of 100% is in sharp con-
trast to all previous reports estimating the incidence of mar-
row micrometastases to be 20–30% in this patient popu-
lation. Because the sensitivity of their assay is within the
same range of that reported with RT-PCR, the question is
raised whether all of these cells were indeed tumor cells.
Other methodologic issues include the specific protocols
regarding how samples are collected and in what volume.
Using a large volume (10mL or perhaps more) may be ideal
for collecting blood samples, particularly in patients who are
on therapy and who may have treatment-related reductions
in the number of circulating mononuclear cells. However,
tions, as demonstrated in a recent report by Helgestad et al.
. They showed that the density of nucleated cells in the
bone marrow of leukemia patients is markedly reduced with
larger volume aspirates, due to potential dilution with pe-
ripheral blood during the collection. In fact, this dilution
effect from larger aspirations resulted in several samples be-
cytometry), despite clearly containing >0.1% tumor cells in
the first small volume sample withdrawn. Further, bilateral
bone marrow aspirations are routinely performed for ES
patients, due to the typically patchy tumor involvement.
Most studies do not specify whether both sides are pooled
together or analyzed separately. Attention to standardization
of collection procedures will help improve interpretation of
Finally, it remains unclear which assay has the greatest
utility. Because of the success of flow cytometry for MRD as-
antibodies, and the encouraging results noted so far in pre-
liminary studies, it is likely that there will be further explo-
ration of flow cytometry for MRD detection in ES. Results
from ongoing trials which directly compare these method-
ologies will hopefully provide input on which assay to study
in larger prospective clinical trials.
Detection of MRD in blood or bone marrow is best estab-
lished for patients with childhood leukemia, where flow cy-
tometry to assess response to therapy is now a standard part
of risk assessment . In adult carcinomas, FDA-approved
methods like the CellSearch assay identify circulating tumor
cells through positive enrichment using epithelial cell-spe-
are now widely employed. Among pediatric solid tumors,
there has been considerable work in MRD detection in neu-
roblastoma (reviewed in ), which like ES is characterized
by disease recurrence following complete remission in a sub-
stantial subset of patients. ES appears particularly well suited
for MRD detection due to tumor-specific translocations that
facilitate RT-PCR and FISH detection as well as expression
of tumor-specific cell surface proteins like CD99 that facil-
itate detection by flow cytometry. Studies using RT-PCR
have demonstrated that otherwise occult tumor cells can
indeed be identified at initial diagnosis in the blood and/or
wise localized disease and that such patients generally have
inferior outcome. Smaller studies have shown that return of
ically apparent relapse and that MRD assessment can be used
to follow response to chemotherapy regimens. Although
effective therapeutic interventions for these findings may not
yet be available in some cases, the results to date support
the contention that clinically meaningful information can be
study is indicated.
While the aforementioned studies have used RT-PCR,
flow cytometry offers a commercially available, less labor-in-
tensive approach with similar sensitivity that may be more
widely applicable, given that detection does not require prior
knowledge of the particular chromosomal translocation.
Also, this method may be less susceptible to degradation of
sample integrity if overnight shipping to a central laboratory
is required. However, further validation in additional studies
is required, and standardization of sample collection, testing
methods, and reporting of results will be critical. Trials are
currently underway which will compare these modalities to
each other, and to compare MRD test results with imaging
studies and overall outcome to further define the overall
utility and clinical relevance of MRD assessment in this dis-
 R. C. Chan, W. W. Sutow, R. D. Lindberg et al., “Management
and results of localized Ewing’s sarcoma,” Cancer, vol. 43, no.
3, pp. 1001–1006, 1979.
 G. Schleiermacher, M. Peter, O. Oberlin et al., “Increased risk
tasis and circulating tumor cells in localized Ewing tumor,”
Journal of Clinical Oncology, vol. 21, no. 1, pp. 85–91, 2003.
 B. Thomson, D. Hawkins, J. Felgenhauer, and J. P. Radich,
“RT-PCR evaluation of peripheral blood, bone marrow and
peripheral blood stem cells in children and adolescents un-
dergoing VACIME chemotherapy for Ewing’s sarcoma and
alveolar rhabdomyosarcoma,” Bone Marrow Transplantation,
vol. 24, no. 5, pp. 527–533, 1999.
 U. H. Athale, S. A. Shurtleff, J. J. Jenkins et al., “Use of
reverse transcriptase polymerase chain reaction for diagnosis
and staging of alveolar rhabdomyosarcoma, Ewing sarcoma
family of tumors, and dsm,oplastic small round cell tumors,”
Journal of Pediatric Hematology/Oncology, vol. 23, no. 2, pp.
 S. Avigad, I. J. Cohen, J. Zilberstein et al., “The predictive
potential of molecular detection in the nonmetastatic Ewing
family of tumors,” Cancer, vol. 100, no. 5, pp. 1053–1058,
 E. de Alava, M. D. Lozano, A. Patino et al., “Ewing family tu-
merase chain reaction detection of minimal residual disease
in peripheral blood samples,” Diagnostic Molecular Pathology,
vol. 7, no. 3, pp. 152–157, 1998.
 M. J. Absalon, M. B. McCarville, T. Liu, V. M. Santana, N.
C. Daw, and F. Navid, “Pulmonary nodules discovered during
the initial evaluation of pediatric patients with bone and soft-
tissue sarcoma,” Pediatric Blood and Cancer, vol. 50, no. 6, pp.
 F. G. Barr and W. H. Meyer, “Role of fusion subtype in Ewing
sarcoma,” Journalof clinicaloncology, vol. 28,no. 12,pp. 1973–
 M. Peter, H. Magdelenat, J. Michon et al., “Sensitive detection
of occult Ewing’s cells by the reverse transcriptase-polymerase
chain reaction,” British Journal of Cancer, vol. 72, no. 1, pp.
 D. Sumerauer, A. V´ ıcha, H. Kucerova et al., “Detection of
minimal bone marrow infiltration in patients with localized
and metastatic Ewing sarcoma using RT-PCR,” Folia Biologica,
vol. 47, no. 6, pp. 206–210, 2001.
 C. Fagnou, J. Michon, M. Peter et al., “Presence of tumor cells
in bone marrow but not in blood is associated with adverse
prognosis in patients with Ewing’s tumor. Societe Francaise
d’Oncologie Pediatrique,” Journal of Clinical Oncology, vol. 16,
no. 5, pp. 1707–1711, 1998.
 D. C. West, H. E. Grier, M. M. Swallow, G. D. Demetri, L.
Granowetter, and J. Sklar, “Detection of circulating tumor
neuroectodermal tumor,” Journal of Clinical Oncology, vol. 15,
no. 2, pp. 583–588, 1997.
 C. Pfleiderer, A. Zoubek, B. Gruber et al., “Detection of tum-
our cells in peripheral blood and bone marrow from Ewing
tumour patients by RT-PCR,” International Journal of Cancer,
vol. 64, no. 2, pp. 135–139, 1995.
 A. Zoubek, R. Ladenstein, R. Windhager et al., “Predictive
potential of testing for bone marrow involvement in Ewing
tumor patients by RT-PCR: a preliminary evaluation,” Inter-
national Journal of Cancer, vol. 79, no. 1, pp. 56–60, 1998.
netic purging of Ewing’s sarcoma from blood and bone mar-
row: quantitation by real-time polymerase chain reaction,”
Journal of Clinical Oncology, vol. 19, no. 16, pp. 3649–3659,
 I. Yaniv, I. J. Cohen, J. Stein et al., “Tumor cells are present
sistence following transplantation is associated with relapse,”
Pediatric Blood and Cancer, vol. 42, no. 5, pp. 404–409, 2004.
 R. S. Bridge, V. Rajaram, L. P. Dehner, J. D. Pfeifer, and
A. Perry, “Molecular diagnosis of Ewing sarcoma/primitive
neuroectodermal tumor in routinely processed tissue: a com-
parison of two FISH strategies and RT-PCR in malignant
8 Sarcoma Download full-text
round cell tumors,” Modern Pathology, vol. 19, no. 1, pp. 1–
 I. Y. Cheung, Y. Feng, K. Danis et al., “Novel markers of
subclinical disease for Ewing family tumors from gene expres-
sion profiling,” Clinical Cancer Research, vol. 13, no. 23, pp.
M. Salzer-Kuntschik, “MIC2 is a specific marker for Ewing’s
sarcoma and peripheral primitive neuroectodermal tumors:
evidence for a common histogenesis of Ewing’s sarcoma and
peripheral primitive neuroectodermal tumors from MIC2
expression and specific chromosome aberration,” Cancer, vol.
67, no. 7, pp. 1886–1893, 1991.
 S. G. Dubois, C. L. Epling, J. Teague, K. K. Matthay, and E.
Sinclair, “Flow cytometric detection of Ewing sarcoma cells in
peripheral blood and bone marrow,” Pediatric Blood and
Cancer, vol. 54, no. 1, pp. 13–18, 2010.
 L. Yang, J. C. Lang, P. Balasubramanian et al., “Optimization
of an enrichment process for circulating tumor cells from the
blood of head and neck cancer patients through depletion of
normal cells,” Biotechnology and Bioengineering, vol. 102, no.
2, pp. 521–534, 2009.
 S. Ash, D. Luria, I. J. Cohen et al., “Excellent prognosis in a
subset of patients with Ewing sarcoma identified at diagnosis
by CD56 using flow cytometry,” Clinical Cancer Research, vol.
17, no. 9, pp. 2900–2907, 2011.
 L. J. Gardner, J. M. Polski, R. Fallon, and C. H. Dunphy,
“Identification of CD56 and CD57 by flow cytometry in Ew-
ing’s sarcoma or primitive neuroactodermal tumor,” Virchows
Archiv, vol. 433, no. 1, pp. 35–40, 1998.
 M. J. Borowitz, M. Devidas, S. P. Hunger et al., “Clinical
significance of minimal residual disease in childhood acute
lymphoblastic leukemia and its relationship to other prognos-
tic factors: a Children’s Oncology Group study,” Blood, vol.
111, no. 12, pp. 5477–5485, 2008.
of FISH, RT-PCR, and RQ-PCR for monitoring the BCR-ABL
gene after hematopoietic stem cell transplantation in CML,”
European Journal of Haematology, vol. 68, no. 5, pp. 272–280,
 J. Helgestad, S. Rosthoj, P. Johansen, K. Varming, and E.
Ostergaard, “Bone marrow aspiration technique may have an
impact on therapy stratification in children with acute lym-
phoblastic leukaemia,” Pediatric Blood and Cancer, vol. 57, no.
2, pp. 224–226, 2011.
 W. J. Allard, J. Matera, M. C. Miller et al., “Tumor cells cir-
culate in the peripheral blood of all major carcinomas but not
in healthy subjects or patients with nonmalignant diseases,”
Clinical Cancer Research, vol. 10, no. 20, pp. 6897–6904, 2004.
 K. Beiske, P. F. Ambros, S. A. Burchill, I. Y. Cheung, and K.
Swerts, “Detecting minimal residual disease in neuroblastoma
patients-the present state of the art,” Cancer Letters, vol. 228,
no. 1-2, pp. 229–240, 2005.