JOURNAL OF PATHOLOGY, VOL.
178: 116--121 (1996)
REVIEW ARTICLE
THE MOLECULAR PATHOLOGY OF SMALL
ROUND-CELL TUMOURS-RELEVANCE TO
DIAGNOSIS, PROGNOSIS, AND CLASSIFICATION
AIDAN P. MCMANUS*, BARRY A. GUSTERSON?, C. ROSS PINKERTON* AND JANET M. SHIPLEY7
Sei t i o i i r of * Paediatt icr and 7 Cell Biology and Experimental Patholog),
INTRODUCTION
The term ‘small round-cell tumour’ (SRCT) tradition-
ally describes a group of undifferentiated paediatric
tumours that can present diagnostic difficulty because
they may be indistinguishable using light microscopy.’
These tumours, which include neuroblastoma, the
Ewing family of tumours, and rhabdomyosarcoma, rep-
resent about 15 per cent of all childhood cancers.2
Classical light microscopic, electron microscopic, and
immunohistochemical features can be highly suggestive
of the tumour type, but in certain cases a definite
diagnosis remains difficult. A precise histological diag-
nosis was of less relevance when treatment simply
involved resection and radiotherapy, but the continued
development of disease-specific therapeutic strategies,
and the concomitant improvement in prognosis, has
rendered accurate tumour diagnosis and classification of
paramount importance.’
It has been clear for some time that the SRCTs exhibit
highly characteristic cytogenetic abnormalities. Increas-
ingly, the identification of these lesions is recognized as
an invaluable aid in elucidating tumour type, but limi-
tations exist in the ability routinely to karyotype these
tumours. These include the requirement for fresh
tumour material, the frequent overgrowth of normal
stronial cells during culture, and the difficult and time-
consuming nature of accurate karyotyping of poor qual-
ity metaphases. These difficulties are compounded by the
increased utilization of primary chemotherapy and mini-
mally invasive biopsy methods. Successful cytogenetic
analysis has been performed in less than 50 per cent of
, ~ typically in less than 30
cases of Ewing’s ~ a r c o m aand
per cent of neuroblastomas.’
The recent molecular genetic characterization of the
chromosome abnormalities characteristic of the SRCTs
has resulted in greatly improved detection strategies,
based mainly on the techniques of fluorescence in situ
hybridization (FISH) and the polymerase chain reaction
(PCR). These are applicable to small amounts of tumour
material. Unique phenotypic markers have also been
Addressee for correspondence: Aidan P. McManus, Molecular
Cytogenetics, F Block, Institute of Cancer Research, 15 Cotswold
Road. Sutton, Surrey SM2 5NG, U.K.
CCC 0022-34 171961020I 16-06
((> 1996 by John Wiley & Sons, L,td.
Institute of Cunter R t x m c h , Sutton, Surrey, U K
identified, which are not only useful diagnostic aids, but
which exhibit specific associations with the chromosome
changes in the SRCTs. These complementary novel
approaches to diagnosis have implications for prognos-
tication and tumour classification. This review outlines
these recent advances and their impact on the diagnosis,
prognosis, and classification of the SRCTs.
THE EWING FAMILY OF TUMOURS
Ewing’s sarcoma is the second most common malig-
nant bone tumour of childhood, but also occurs as a
primary soft tissue neoplasm without involvement of
bone. Histologically, the tumour cells are uniformly
bland and undifferentiated, with a relatively low mitotic
index.6 This tumour is closely related to a diverse group
of small round-cell tumours collectively referred to
as ‘peripheral primitive neuroectodermal tumours’
(pPNETs), which occur outside the autonomic nervous
system and generally exhibit features of neuroecto-
dermal differentiati~n.~Terms including ‘peripheral
neuroepithelioma’, ‘Askin tumour’, and ‘primitive
neuroectodermal tumour’ (PNET) are used, often inter-
changeably, creating a confused nosology. pPNET and
Ewing’s sarcoma are characterized by a highly recurrent
chromosome translocation, t( I 1;22)(q24;~12),~ and the
expression of the glycoprotein ~30132,encoded by the
MIC2 gene.* The identification of these features has
not only aided diagnosis, but has also strengthened
the concept that these tumours are part of a disease
spectrum, forming a Ewing family of t u m ~ u r s . ~
The t( 11;22)(q24;q12) has been fully characterized and
the genes disrupted by the translocation have been
cloned. The translocation results in the production of a
chimeric gene between EWS, a novel putative RNA-
binding gene located at 22q12, and F,!,Il,’o a member of
the ETS family of transcription factors located at 1 lq24.
The resulting fusion transcript has transforming activity
and has therefore been implicated in pathogenesis. ’’
Two variant translocations have been described and
characterized. The t(21;22)(q12;ql2) involves a gene
on chromosome 21 known as E R I S , ’ ~ , ’and ~
t(7;22)(p22;q12) a gene, ETVZ, at 7 ~ 2 2 ,both
’ ~ members
of the same family of ETS genes.
 THE MOLECULAR PATHOLOGY OF SMALL ROUND-CELL TUMOURS
The molecular genetic characterization of
t( I 1 ;22)(q24;q 12) has facilitated novel approaches to
translocation detection. FISH is a relatively simple,
non-isotopic slide-based technique that involves hybrid-
izing DNA probes, specific to the chromosomal regions
of interest, to cell preparations and visualizing their
location using fluorescent reporter molecules. Cosmid
probes, by virtue of their defined and specific hybrid-
ization pattern, are particularly useful in detecting
structural chromosome abnormalities in interphase
cell populations. Interphase FISH to detect
t(11;22)(q24;q12) was first reported in 1992l5*I6and its
use as a diagnostic tool was subsequently ~ e r i f i e d . In
'~
1994, this was further refined by the description of
cosmids closely flanking the breakpoints;" their use in
interphase FISH as a rapid and reliable method of
diagnosis using tumour touch imprints was subsequently
described. l9
The t(l1;22)(2q4;q12) can also be identified by detec-
tion by reverse transcription PCR (RT-PCR) of the
fusion gene transcript produced by the translocation.
This is dependent on the availability of viable RNA and
is based on the fact that the chromosome translocation
results in the formation of a fusion gene, composed of a
part of each gene located at the chromosome break-
points. RNA is copied by the enzyme reverse tran-
scriptase into complementary DNA (cDNA); with an
appropriate primer from each of the two genes, the
specific fusion gene can then be amplified by PCR. Only
material containing the tumour-specific fusion gene will
give a positive PCR amplification, which can be visual-
ized as a discrete band of defined size on an agarose gel.
RT-PCR has been used to detect the fusion transcript
formed by t(11;22)(q24;q12) in over 100 cases of the
Ewing tumour family. It was first reported with the
cloning of the genes" and several publications have
subsequently described the detection of fusion tran-
scripts as a diagnostic aid.'2320,2'In 1994, analysis of 114
sarcomas revealed the presence of a fusion transcript in
95 per cent of cases carrying the diagnosis of Ewing's
sarcoma or pPNET, confirming the diagnostic utility
of RT-PCR. It was suggested that the presence of the
specific fusion transcript defines a subgroup of SRCTs-
the Ewing family of tumours.22
Several subtly different E WSIFLIl and E WSIERG
fusion transcripts have been described. In their analysis
of 89 Ewing family tumours, Zucman et ul. described 13
different fusions and predicted that these would result in
the production of a similar protein in all cases. It was
suggested, therefore, that the exact nature of the chro-
mosome breakpoint was probably irrelevant to the
potential oncogenic behaviour of the protein.12 Others
have suggested that these different transcripts may be
associated with different biological behaviours, but all
studies have so far failed to detect significant correla-
tions between different chimaeric E WS transcripts and
any clinical parameters.22,2'
The cell surface glycoprotein p30/32M1C2is highly, but
not exclusively, expressed in the Ewing family of
tumours.8 It is encoded by the MIC2 gene, located in the
pseudo-autosomal region of the X and Y chromo-
s o m e ~and ~ ~is thought to perform a role in cellular
1 I7
adhesion processes.25 Several monoclonal antibodies
which detect different epitopes of this antigen have been
developed; most studies have employed HBA71 or 0 1 3
in the analysis of Ewing tumours. Up to 98 per cent of
the Ewing family of tumours exhibit immunoreactivity
with HBA71, but non-neoplastic tissues and other
tumour types can occasionally give positive results,
including alveolar rhabdomyosarcoma, embryonal
rhabdomyosarcoma, and non-Hodgkin's lymphoma.
However, the positive staining pattern identified in a
small subset of rhabdomyosarcoma cases is different
from that observed in Ewing cases; only a small number
of cells show a heterogeneous staining pattern, as
opposed to the diffuse and widespread positivity
observed in Ewing cases. Non-neoplastic tissues that can
react include smooth and skeletal muscle cells and
subsets of bone marrow, lymph nodal, and splenic
From the limited number of studies employing 013,
the largest of these examined the immunostaining char-
acteristics of 21 Ewing tumours and 147 other tumours,
including tumours which form part of the differential
diagnosis of Ewing tumours.8 0 1 3 was shown to be 100
per cent sensitive for Ewing tumours. No staining was
evident in cases of neuroblastoma, rhabdomyosarcoma,
and non-lymphoblastic lymphoma, but limited immuno-
reactivity was shown in a small number of non-Ewing
tumours, including lymphoblastic lymphoma. The
authors concluded that within the context of a diagnos-
tic laboratory where other immunophenotypic informa-
tion was available, immunoreactivity with 0 1 3 should
be considered very good evidence that a tumour belongs
to the Ewing family. 0 1 3 can be employed on formalin-
fixed, paraffin-embedded tissue sections and on decalci-
fied material."
The association between t(l1;22)(q24;q12), E WS gene
rearrangement, and the expression of MZC2, assessed by
both HBA71 and 0 1 3 antibodies, was analysed in a
group of Ewing's sarcoma and PNET cases.27Nineteen
of 20 cases exhibited positive immunoreactivity using
both antibodies. The one case that did not express MIC2
possessed a variant t(l1;22). Three of the cases which
were positive for MIC2 did not show evidence of EWS
involvement by Southern blot analysis. The authors
indicated the importance of combining diagnostic
modalities in the complete evaluation of this tumour
type.
The prognostic relevance of grouping Ewing's sar-
coma and PNETs into a Ewing family of tumours is
unclear.28 Poor prognosis has been associated with
an increasing degree of neuroectodermal differentia-
ti01-1.~~Comparisons of Ewing's sarcoma with PNET
cases in the German tumour registry revealed a poorer
outcome in cases of PNET.30 However, recent trials
employing intensive multi-agent chemotherapy regi-
mens suggest that the survival of PNET patients is
comparable to that of cases of bone or soft-tissue
Ewing's sarcoma of a comparable The results
from the current national inter-group studies in Europe
and the U.S.A. may provide important insight into the
prognostic worth of grouping these tumours into a
single family.
118 A. P. McMANUS ET AL.
NEUROBLASTOMA
Neuroblastoma originates from the sympathoadrenal
lineage of the neural crest34 and is the most common
extra-cranial tumour of childhood. The disease exhibits
a complex clinical evolution, showing different degrees
of aggressi~eness,~’ mirrored by heterogeneous biologi-
cal characteristics. Cytogenetic studies have revealed
characteristic chromosome changes which not only sup-
port the diagnosis, but which correlate with disease stage
and help to predict tumour behaviour. These aberra-
tions are deletions of the short arm of chromosome 1 (at
1p36.2-3), amplification of the proto-oncogene M YC N ,
and abnormalities of the chromosome number
(ploidy).36More recently, the roles of the neurotrophins
and their receptors, a family of related molecules that
promote neuronal survival and differentiation in the
central and peripheral nervous system,37 have been
examined in neuroblastoma and a novel prognostic
marker, TRKA gene expression, has been identified.38
Deletion of l p is the most common cytogenetic abnor-
mality in neuroblastoma.” The consensus deletion has
been mapped very precisely to the distal end of the short
arm of chromosome 1 at p36.2-3 and it has been
proposed that either one or two putative tumour-
suppressor genes lie in this r e g i ~ n ,but
~ ~ so
. ~far
~ none
has been isolated. Cytogenetic evidence of gene ampli-
fication, in the form of extrachromosomal double
minutes, or homogeneously staining regions, has been
recognized for some time in both primary tumours and
cell lines; the amplified sequences have been shown to
include MYCN.41 Virtually all of these cases have high
MYCN expression at the RNA and protein levels.42 A
strong correlation between MYCN amplification and
loss of l p material has been demonstrated in some
studies.43Both MYCN and l p loss correlate with poor
outcome and with each other.44 These genetic lesions
have been identified by the International Neuroblastoma
Staging System and Response Criteria Committee as
important biological factors which should be measured
pro~pectively.~’
Interphase FISH analysis on tuniour touch imprints
and bone marrow smears has been shown by Taylor
et al. to be an effective way to identify the specific cyto-
genetic changes associated with neuroblastoma.’ Using
a chromosome 1 probe specific for the heterochromatic
region at lq12, a cosmid probe specific for the consensus
deletion at lp36, and a MYCN plasmid probe, they
reported the detection of Ip deletions and MYCN
amplification and estimated the tumour ploidy in six
cases of neuroblastoma. These results were confirmed by
cytogenetics in four of the cases. At least one study has
described detection of 1 deletions in paraffin sections of
neuroblastoma tissue.“RT-PCR assays to detect over-
expression of M Y C N as a result of amplification have
also been de~cribed.~’-~’
The expression of the proto-oncogene TRKA, which
forms the primary component of the nerve growth factor
receptor, has been shown to be a powerful prognostic
marker that identifies a favourable group of tumours. 3’
The expression of TRKA correlates with favourable
tumour stage and is essentially absent in neuroblastomas
with MYCN amplification. Univariate analysis has
revealed that TRKA expression levels and MYCN copy
number are the most powerful predictors of out~ome.~’
It has recently been proposed that neuroblastomas
may be classified into three genetic subgroups based
on their cytogenetic and molecular profile.49 Type 1
tumours are hyperdiploid or triploid, rarely show struc-
tural chromosome changes, do not display MYCN
amplification, and normally have high TRKA expres-
sion. These patients are likely to be infants with low-
stage disease and a very favourable prognosis. Type 2
tumours are diploid or tetraploid, frequently have l p
deletions, do not exhibit M YCN amplification, and
generally have absent TRKA expression. These patients
have advanced-stage disease with an intermediate prog-
nosis. Type 3 patients are similar to type 2, but have
MYCN amplification. Their prognosis is exceptionally
poor. This classification identifies neuroblastoma as a
group of related tumours that can be segregated into
clinically distinct groups on the basis of their genetic
profile.
DESMOPLASTIC SMALL ROUND-CELL
TUMOUR
Intra-abdominal desmoplastic small round-cell
tumour (DSRCT) is a recently recognized clinicopatho-
logical entity. This extremely rare, highly aggressive
polyphenotypic malignancy exhibits a nesting growth
pattern, prominent desmoplasia, and immunohisto-
chemical reactivity for epithelial, neural, and muscle
markers.” Few karyotypic studies have been performed
on DSRCT, but t(l1;22)(p13;q12) has consistently been
The molecular characterization of the
breakpoint regions has revealed the creation of a fusion
gene between the E W S gene and the Wilms’ tumour 1
gene ( WT1).53
No interphase FISH studies detecting t(l1;22)
(p13;q12) have been described, but RT-PCR has been
reported as a diagnostic tool in two report^.^^,'^ In total,
six tumours regarded as DSRCT and one described as
an undifferentiated carcinoma of unknown primary
site55have exhibited an EWSI WTI fusion transcript by
RT-PCR.
RHABDOMYOSARCOMA
Rhabdomyosarcomas are clinically aggressive skeletal
muscle-derived tumours that usually present in child-
hood and are the most common soft tissue sarcomas in
children6 Their light microscopic diagnosis is based on
the identification of rhabdomyoblasts, but they exhibit a
spectrum of differentiation from primitive mesenchymal
cells to fetal myotubes. Poorly differentiated forms are
common; these are not only difficult to distinguish from
other small round-cell tumours, but are hard to classify
as either embryonal or alveolar, the two main histologi-
cal patterns recognized in rhabdomyosarcoma.s6 Scrable
et al. were the first to suggest that evidence of commit-
ment to the myogenic lineage by detection of muscle-
 THE MOLECULAR PATHOLOGY OF SMALL ROUND-CELL TUMOURS
specific gene expression, and the ascertainment of
specific genotypic changes, could be used in the diagno-
sis and classification of rhabdomy~sarcomas.~~ The
translocation t(2; 13)(q35;q14) has consistently been
detected in the alveolar form of r h a b d o m y o s a r c ~ m a . ~ ~ of the gene PAX3 (located at 2q35), a developmentally
The more common embryonal form, although not
exhibiting a consistent cytogenetic profile, demonstrates
loss of heterozygosity (LOH) on the short arm of
chromosome 11, at 1 1 ~ 1 5 . 5 . ~ ~
The commitment to the myogenic lineage is under the
control of a small set of regulatory proteins.59 These
proteins include MyoD1, a nuclear phosphoprotein
expressed during normal skeletal muscle myogenesis
which performs a critical role in commitment to myo-
genesis.60 MyoDl expression has not been detected in
normal adult tissue, but is expressed in rhabdomyosar-
comas and in the myogenic component of Wilms'
tumour and ectomesenchymoma. It is not expressed in
other tissues or soft tissue sarcomas.61 The utility of
detecting MyoDl gene expression both at the RNA and
at the protein level as a diagnostic aid in rhabdomyosar-
comas has been assessed in several studies. Northern
blot analysis of RNA from two large series of rhab-
domyosarcomas and other paediatric tumours revealed
that MyoDl expression was unique to rhabdomyosar-
The potential of MyoDl expression as a
diagnostic tool has been expanded by the development
of a polyclonal antiserum and a monoclonal antibody,
5.8A, to the MyoD1 protein. Their use in the differential
diagnosis of rhabdomyosarcoma has been demonstrated
in large series of paediatric solid turn our^.^^^^^ This
antibody has been further employed in the study of adult
soft tissue sarcomas. It was found to be extremely
sensitive and s ecific in its ability to detect muscle
This has helped to confirm the exist-
ence of pleomorphic rhabdomyosarcoma, a rare adult
form of rhabdomyosarcoma whose description has been
Embryonal rhabdomyosarcoma appears exclusively
to exhibit LOH at llp15.5. In their analysis of 60
paediatric tumours, Scrable et al. demonstrated that this
LOH was specific to the embryonal form and was
present in 13 of 14 embryonal tumours a n a l y ~ e dA .~~
candidate gene located at 1 lpl5.5-the insulin-like
growth factor-I1 gene (IGF-11)-has been identified.
This gene exhibits genomic imprinting, a phenomenon
whereby gene ex ression is determined specifically by its
parental origin6 IGF-I1 is exclusively silent at the
maternal allele in normal human tissue, but is highly
expressed in rhabdomyosarcoma. Interestingly, overex-
pression of this gene has been implicated as the causative
gene in Beckwith-Wiedemann syndrome (BWS). Spo-
radic BWS results from inheritance of both IGF-I1
alleles from the father and predisposes to rhabdomyo-
sarcoma.67A recent study has shown that imprinting of
IGF-I1 is lost in rhabdomyosarcoma, with IGF-I1 being
transcribed biallelically. Of further interest was the
demonstration that in one case of embryonal rhabdo-
myosarcoma, heterozygosity, but not imprinting, was
retained at the IGF-I1 locus, suggesting that loss of
imprinting may be the functional equivalent of LOH
and may result in the overexpression of IGF-II.68
119
Numerous cytogenetic studies have identified
t(2; 13)(q35;q14) consistently in the alveolar form." The
molecular consequence of the t(2; 13)(q35;q14) has been
fully elucidated. This results in the fusion of 5' sequences
regulated transcription factor involved in muscle devel-
~ p m e n t , ~to' 3' sequences of the gene FKHR (located at
13q14),71identified as a member of the fork head family
of transcription factors. The PAX3-FKHR fusion pro-
tein created by the translocation has been shown to be a
more potent transcriptional activator than PAX3 pro-
tein alone, implicating the translocation product in
p a t h ~ g e n e s i sA
. ~variant
~ t( 1 ;13)(p36;q14) translocation
has been described and characterized, involving PAX7
on chromosome 1, a member of the same family of PAX
genes.73 Both interphase FISH and RT-PCR studies of
rhabdomyosarcoma have been reported.
Two studies recently described the development of
interphase FISH for detection of t(2; 13)(q35;q14).Biegel
et ul. examined the cytogenetic and interphase FISH
profile of 14 small round-cell tumour cell lines and 20
clinical cases of rhabdornyosar~oma.~~ All ten cases of
alveolar histology exhibited PAX3-FKHR fusions and
eight of the cases were regarded as tetraploid. None of
the embryonal tumours exhibited the translocation, but
trisomy 2 was detected in nine of ten cases. McManus
et ul. described the development of a similar interphase
FISH method and demonstrated its utility in assess-
ing minimally invasive biopsies for the presence of
t(2;13)(q35;q14).75
RT-PCR for t(2;13)(q35;q14) was first re orted with
the cloning of the genes at the breakpoint^.^' Two large
studies have subsequently examined series of rhabdomy-
osarcomas for the presence of PAX3-FKHR and PAX7-
FKHR fusion transcripts. In total, 42 of 49 alveolar
rhabdomyosarcomas and 4 of 42 embryonal rhabdomy-
osarcomas demonstrated a fusion t r a n ~ c r i p t .It~ ~was ,~~
also shown that multiplex RT-PCR, performed by com-
bining primers for PAX3IFKHR and EWSIFLII in a
single RT-PCR reaction, could detect the fusion tran-
scripts resulting from either the t(l1;22)(q24;q12) or the
t(2;13)(q35;q14).76
Alveolar histology is found in 20 per cent of rhab-
domyosarcomas and is an adverse prognostic
These patients often present in adolescence and the
tumours are frequently associated with distant metas-
tases. Even with localized disease the outcome is poorer
than with the embryonal type.77378 There is now a trend
towards treating low-stage tumours with more intensive
chemotherapy if there is evidence of alveolar histology,
which is supported by the detection of t(2; 13)(q35;q14).
A 'solid variant' form of alveolar rhabdomyosarcoma,
lacking an alveolar growth pattern by cytologically
similar to the alveolar form, has been recognized in the
NCI classification; this has been shown to exhibit
t(2;13)(q35;q14) and to have a similarly poor progno-
s ~ sIn. ~ certain
~ cases, tumours previously regarded as
embryonal, but exhibiting t(2; 13)(q35;q14) have been
reclassified as solid alveolar rhabdomyosar~omas.~~ The
prognostic value of ploidy, assessed by measuring
tumour DNA content, was recently examined in 34 cases
of embryonal rhabdomyosarcoma." Hyperdiploidy was
120 A. P. McMANUS ET AL.
associated with an excellent prognosis, whereas diploidy
was associated with a poor outcome. From an examin-
ation of the molecular cytogenetic data published so far,
interphase FISH suggests that alveolar rhabdomyo-
sarcomas exhibit t(2; 13)(q35;q14) in pseudodiploid or
pseudotetraploid populations, and that embryonal rhab-
domyosarcomas are consistently hyperdiploid, with tri-
somy of chromosome 2.74*75Considering that cases
regarded as embryonal type have exhibited PAX3-
FKHR fusion transcripts by RT-PCR analysis,54further
studies are required to define the association between
ploidy, t(2;13)(q35;q14), and histology, and to help
refine risk-directed therapy for rhabdomyosarcoma.
CONCLUSION
Substantial improvements have been made in the
treatment and survival of children with SRCT, resulting
in an increased emphasis on precise histological diag-
nosis. Although diagnostic procedures such as electron
microscopy and immunocytochemistry contribute in
poorly differentiated cases, an accurate diagnosis can
remain elusive in a proportion of SRCTs. The cytoge-
netic and molecular genetic abnormalities characteristic
of the different SRCTs can now be consistently and
rapidly identified from minimal quantities of tumour
material, using the techniques of FISH and PCR. This,
coupled with the identification of novel phenotypic
characteristics, has had a major impact on SRCT
diagnosis.
The aim of a tumour classification is to identify
disease entities which are biologically distinct and whose
recognition is of clinical value. The recent advances
described above demonstrate that the SRCTs are geno-
typically and phenotypically distinct tumour types and
that the genetic abnormalities represent key alterations
that influence both the morphology and the clinical
behaviour of the tumour. This suggests that these
advanced phenotypic and genotypic analyses should
form an integral and complementary part of the labora-
tory assessment and clinical management of these forms
of paediatric cancer.
REFERENCES
I. Variend S. Small cell tumours in childhood: a review. J Puthol 1985; 145
1-25.
2 . Stiller C. Aetiology and epidemiology. In: Plowman PH, Pinkerton CR,
eds. Paediatric Oncology: Cliiilcal Practice and Controversies. London:
Chapman Hall, 1992; 1-24.
3. Pinkerton CR, Pritchard-Jones K, Carter RL, Cooper C, McManus AP.
Small round-cell tumours of childhood. Lancet 1994; 344: 725-729.
4. Douglass EC. Chromosomal rearrangements in Ewing’s sarcoma and
peripheral neui-oectoderinal tumor (PNET). Semin Dev Bud 1990; 1: 393-
396.
5. Taylor CPF, McGuckiii AG, Bown NP, Reid MM, Malcolm AJ. Rapld
detection of prognostic genetic factors in neuroblastoma using fluorescence
in .\itu hybridisation on tumour imprints and hone marrow. BY J Cancer
1994; 6 9 445-451
6. Enzinger FM, Weiss SW. Soft Tissue Tumors. 3rd edn. Missouri: Moshy-
Year Book, 1995.
7. Jurgens HF. Ewing’s sarcoma and peripheral primitive neuroectodermal
tumor. Curr Opin O n d 1994; 6 391 396.
8. Weidner N. Tjoe J. Immunohistochemical profile of monoclonal antibody
013: antibody that recognizes glycoprotein ~ 3 0 / 3 2 and~ ’ ~is ~useful in
diagnosing Ewing’s sarcoma and peripheral neuroepithelioma. Am J Surg
Pafhoi 1994: IS:486494.
9. Horowitr ME, Malawer MM, Delaney TF, Tsokos MG. Ewing’s sarcoma
family of tumors: Ewing’s sarcoma of bone and the peripheral primitive
neuroectodermal tumors. In: Pizzo PA, Poplacks DG, eds. Principles and
Practice of Paediatric Oncology. Philadelphia: J. B. Lippincott, 1993;
795-821.
10. Delattre 0, Zucman J, Plougastel B, ef ul. Gene fusion with an ETS
DNA-binding domain caused by chromosome translocation in human
tumours. Nurure 1992; 359 162-165.
I I . Ohno T, Rao VN, Reddy ESP. EWSIFLI-1 chimeric protein is a transcrip-
tional activator. Cunccr Res 1993; 53: 5859-5863.
12. Zucman J, Melot T, Desmaze C, ef ul. Combinatorial generation of variable
fusion proteins in the Ewing family o f tumonrs. EMBO J 1993; 12:
448 14487.
13. Dunn T, Praissman L, Hagag N, Viola MV. ERG gene is translocated in an
Ewing’s sarcoma cell line. Cuncer Gene1 Cytogenet 1994; 7 6 19-22.
14. Jeon IS, Davis JN, Braun BS, et a/. A variant Ewing’s sarcoma translo-
cation (7;22) fuses the EWS gene to the ETS gene ETVI. Oncogene 1995; 1 0
1229-1234.
15. Giovannini M, Selleri L, Biegel JA, Scotlandi K, Emanuel BS, Evans GA.
Interphase cytogenetics for the detection of the t(l1;22)(q24;q12) in small
round cell turnours. J Clin fnvesr 1992; 9 0 191 1-1918
16. Desmaze C, Zucinan J, Delattre 0, Thomas G, Aurias A. Unicolor and
bicolor in situ hybridisation in the diagnosis of peripheral neuroepithelioma
and related tumors. Gene Chromosome Cuncer 1992; 5: 30-34.
17. Taylor CF, Patel K, Jones T, Kiely F, Stavola BLD, Sheer D. Diagnosis of
Ewing’s sarcoma and peripheral neuroectodermal tuinour based on the
detection of t(l1;22) using fluorescence in J i t u hybridisation. Br J Cunwr
1993; 67: 128-133.
18. Desmaze C, Zucman J , Delattre 0, Thomas G, Aurias A. Interphase
molecular cytogenetics or Ewing’s sarcoma and peripheral neuroepithe-
lioma t(l1;22) with flanking and overlapping cosniid probes. Curicer Gf,ncr
C‘ytogenef 1994; 7 4 13-18.
19. McMaiius AP, Gusterson BA, Pinkerton CR, Shipley JM. Diagnosis of
Ewing’s sarcoma and related tumours by fluorescence tn sittr hybridisation
detection of chromosome 22q12 translocations on tumour touch imprints.
J Puthol 1995; 176 137-142.
20. Downing JR, Head DR, Parham DM, er ul. Detection of the
( I 1;22)(q24;q12) translocation of Ewing’s sarcoma and peripheral neuro-
ectodermal tumor by reverse transcription polymernsc chain rcaction. A m J
Pathol 1993; 1 4 3 1294-1300.
21. Sorensen PHB, Lui XF, Delattre 0, er ul. Reverse transcription PCR
amplification of EWSIFLI-1 fusion transcripts as a diagnostic tcst for
peripheral primitive neuroectodermal tumors of childhood. Duzgn Mol
Pothol 1993;
22. Delattre 0, Zucinan J, Melot T, et a/. The Ewing family of tumors -a
subgroup of small round-cell tumors defined by specific chimeric transcripts.
N Engl J Med 1994; 331: 294-299.
23. Zoubek A, Pfleiderer C, Salzer-Kuntschik M, @f al. Variability of EWS
chimaeric transcripts in Ewing tumours: a comparison of clinlcal and
molecular data. Br J Cuncer 1994; 7 0 908-913.
24. Fellinger EJ, Garin-Chesa P, Sui SL, DeAngelis P. Lane .IM, Rettig WJ.
Biochemical and genetic characterization of the HBA7 1 Ewing’s sarcoma
cell surface antigen. Cancer Res I Y Y I ; 51: 336-340.
25. Gelin C, Aubrit F, Phalipon A, et ul. The E2 antlgen, a 32 kd glycoprotein
involved in T-cell adhesion processes, is the MIC2 genc product. E M B O J
1989; 8: 3253-3259.
26. Amhros IM, Ambros PF. Strehl S, Kovar H, Gadner H , Salzer-Kuntschik
M. MIC2 is a specific marker for Ewing’s sarcoma and peripheral primitive
neuroectodermal tumors: evidence lor a common histogenesis of Ewing’s
sarcoma and peripheral primitive neuroectodermal tumors from MIC2
expression and spccific chromosome aberration. Cuncer I99 I ; 67: I886
1893.
27. Ladanyi M, Lewis R, Garin-Chesa P, et ul. EWS rearrangement in Ewing’s
sarcoma and peripheral neuroectodermal tumor. Molecular detection and
correlation with cytogenetic analysis and MlC2 expression. D i q n Mol
Puthol 1993: 2 141-146.
28. Kretschmar CW. Ewing’s sarcoma and the ‘Peanut’ tumors. N Engl J Med
1994; 331: 325-327.
29. Dehner LP. Primitive neuroectodermal tumor and Ewing’s sarcoma. Ani J
Surg Parhol 1993; 17: 1 13.
30. Schmidt D, Herrmann C, Jurgens H, Harms D. Malignant peripheral
neuroectodermal tumor and its necessary distinction from Ewing’s sarcoma:
a report from the Kiel Pediatric Tumor Registry. Cancer 1991; 68: 2251-
2259.
31. Burdagh S, Jurgens H, Peters C. Myeloablative radiochemotherapy and
hematopoietic stem-cell rescue in poor prognosis Ewing’s sarcoma. J Clin
Oncol 1993; 11: 1482-1488.
32. Jurgens H, Bier V, Harms D, er a/.Malignant peripheral neuroectodermal
tumors: a retrospective analysis of 42 patients. Cancer 1988; 61: 1 4 8 2 ~148X.
33. Miser JS, Kinsella TJ, Triche TJ, et a/.Treatment o f peripheral neuroepi-
thelioma in children and young adults. J Clin Oncol 1987; 5: 1752-1758.
34. Bolande RP. The neurocristopathies: a unifying concept of disease arising in
neural crest maldevelopment. Hum Parhol 1974; 5: 409 429.
35. Tonini GP. Neuroblastoma: a multiple biological disease. Eur J C’uncer.
1993; 29A: 802-804.
 THE MOLECULAR PATHOLOGY OF SMALL ROUND-CELL TUMOURS
36. Brodeur GM, Fong C. Molecular biology and genetics of human neuro-
blastoma. Cuncer Genet Cvrozenet 1989: 41: 153-174.
37. Glass DJ, Yancopoulos GD. The neurotrophins and their receptors. Trends
Cell B i d 1993; 3: 262-268.
38. Nakagawara A, Arima-Nakagawara M, Scavarda NJ, Azar CG, Cantor
AB, Brodeur GM. Association between high levels of expression of the
TRK gene and favourable outcome in human neuroblastoma. N Engl J Med
1993; 3 2 8 847-854.
39. Takeda 0, Homma C, Maseki N, ef al. There may be two tumor suppressor
genes on chromosome arm l p closely associated with biologically distinct
subtypes of neuroblastoma. Gene Chromo.same Cancer 1994; 1 0 30-39.
40. Schleiermacher G, Peter M, Michon J, er al. Two distinct deleted regions on
the short arm of chromosome 1 in neuroblastoma. Gene Chrumosome
Cancer 1994; 1 0 275-281.
41. Brodeur GM, Seeger RC. Gene amplification in human neuroblastoma:
basic mechanisms and clinical implications. Cancer Genet Cyrogenet 1986;
1 9 101-111.
42. Slavc I, Ellenbogen R, Jung WH, er ul. myc gene amplification and
expression in primary human neuroblastoma. Cuncer Rrs 1990; 5 0 1459-
1463.
43. Fong CT, White PS, Peterson K, er al. Loss of heterozygosity for chromo-
somes 1 or 14 defines subsets of advanced neuroblastomas. Cancer Rex 1992;
52: 1780-1785.
44. Hayashi Y, Kanda N. Inaba T, ef ul. Cytogenetic findings and prognosis in
neuroblastoma with emphasis on marker chromosome 1. Cancer 1989; 63:
126-132.
45. Brodeur GM, Pritchard J, Berthold F: et ul. Revisions of the international
criteria for neuroblastoma diagnosis, staging, and response to treatment.
J Clin Oncol 1993; 11: 1466-1477.
46. Stock C, Ambros IM, Mann G, Gadner H, Amann G, Ambros PF.
Detection of lp36 deletions in paraffin sections of neuroblastoma tissues.
Gene Chromosome Cancer 1993; 6 1-9.
47. Gilbert J, Norris MD, Haber M, Kavallaris M, Marshall GM, Stewart BW.
Determination of N-myc gene amplification in neuroblastoma by differen-
tial polymerase chain reaction. Mol Cell Probes 1993; 7: 227-234.
48. Boerner S, Squire J, Thorner P, McKeniia G, Zielenska M. Assessment of
MYCN amplification in neuroblastoma biopsies by differential polymerase
chain reaction. Paediutr Parhol 1994; 1 4 823-832.
49. Brodeur GM. Molecular basis for heterogeneity in human neuroblastomas.
Eur J Cancer 1995; 31A: 505-509.
50. Gerald WL, Miller HK, Battiford H, Miettinen M, Silva EG, Rosai J.
Intra-abdominal desmoplastic small round-cell tumor A m J Surg Pathol
1991; 15: 499-513.
51. Sawyer JR, Tryka AF, Lewis JM. A novel reciprocal chromosome trans-
location t(l1;22)(p13;q12) in an intraabdominal desmoplastic small round-
cell tumor. A m J Suvg Parhol 1992; 1 6 411416.
52. Rodriguez E, Sreekantaiah C, Gerald W, Reuter VE, Motzer RJ, Chaganti
RSK. A recurring translocation, t(l1;22)(~13;q12), characterizes intra-
abdominal desmoplastic small round-cell tumors. Cancer Genet Cytogenet
1993; 6 9 17-21
53. Ladanyi M, Gerald W. Fusion of the EWS and WTI genes in the
desmoplastic small round cell tumor. Cancer Res 1994; 54: 2837-2840.
54. Barr FG, Chatten J, DCruz CM, er al. Molecular assays for chromosomal
translocations in the diagnosis of pediatric soft tissue sarcomas. J A m Med
A.PCOC1995; 273 553-557.
55. Motzer RJ, Rodriguez E, Reuter VE, Bosl GJ, Mazumdar M, Chaganti
RSK. Molecular and cytogenetic studies in the diagnosis of patients with
poorly differentiated carcinomas of unknown primary site. J Clin Oncol
1995; 1 3 274-282.
56. Tsokos M, Webber BL, Parham DM, ef ul. Rhabdomyosarcoma-a new
classification scheme related to prognosis. Arch Pathol Lab Med 1992; 116
847-855.
57. Scrable H, Witte D, Shimada H, ei a/. Molecular differential pathology of
rhabdomyosarcoma. Gene Chromusome Cancer 1989; 1: 23-35.
121
58. Turc-Carel C, Lizard-Nacol S, Justrabo E, Favrot M, Philip T, Tabone E.
Consistent chromosomal translocation in alveolar rhabdomyosarcoma.
Cancer Genet Cytogenet 1986; 1 9 361-362.
59. Buckingham M. Which myogenic factors make muscle? Curr Biol 1994; 4
61-63.
60. Davis RL, Weintraub H, Lassar AB. Expression of a single transfected
cDNA converts fibroblasts to myoblasts. Cell 1987; 51: 987-1000.
61. Dias P, Parham DM, Shapiro DN, Webber BL, Houghton PJ. Myogenic
regulatory protein (MyoDl) expression in childhood solid tumors: diagnos-
tic utility in rhabdomyosarcoma. A m J Pathol 1990; 137: 1283-1291
62. Clark J, Rocques PJ, Braun T, t.t al. Expression of members of the myf gene
family in human rhabdomyosarcomas. Br J Cancer 1991; 6 4 1049-1052.
63. Dias P, Parham DM, Shapiro DN, Tapscott SJ, Houghton PJ. Monoclonal
antibodies to the myogenic regulatory protein MyoDI: epitope mapping
and diagnostic utility. Cancer Res 1992; 5 2 6431-6439.
64. Tallini G, Parham DM, Dias P, Cordon-Cardo C, Houghton PJ, Rosai J
Myogenic regulatory protein expression in adult soft tissue sarcomas. A m J
Pathol 1994; 1 4 4 693-701.
65. Wesche WA, Fletcher CDM, Dias P, Houghton PJ, Parham DM. Immuno-
histochemistry of MyoDl in adult pleomorphic soft tissue sarcomas. Am J
Surg Parhol 1995; 1 9 261-269.
66. Barlow DP. Methylation and imprinting: from host defence to gene
regulation. Science 1993; 2 6 0 309-310.
67. Karp JE, Broder S. New directions in molecular medicine. Cancer Res 1994;
5 4 653-665.
68. Zhan S, Shapiro DN, Helman LJ. Activation of an imprinted allele of the
insulin-like growth factor I1 gene implicated in rhabdomyosarcoma. Proc
A A C R 1994; 3 5 3420a.
69. Sandberg AA, Bridge JA. The Cytogenetics of Bone and Soft Tissue
Tumors. Austin: R. G. Landes, 1994.
70. B a n FG, Galili N, Holick J, Biegel JA, Rovera G, Emanuel B. Rearrange-
ment of the PAX3 paired box gene in the paediatric solid tumour alveolar
rhabdomyosarcoma. Nature Gener 1993; 3 113-1 17.
71. Galili N, Davis R, Fredericks W, ef ul. Fusion of a fork head domain gene
to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nature Genet
1993; 5 230-235.
72. Fredericks WJ, Galili N, Mukhopadhyay S, et a/. The PAX3-FKHR fusion
protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas
is a more potent transcriptional activator than PAX3. M u / Cell B i d 1995;
15: 1522-1535.
73. Davis RJ, D’Cruz CM, Lovell MA, Biegel JA, Barr FG. Fusion of PAX7 to
FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdo-
myosarcoma. Cancer Res 1994; 5 4 2869-2872.
74. Biegel JA, Nycum LM, Valentine V, Bar FG, Shapiro DN. Detection of the
t(2;13)(q35;q14) and PAX3-FKHR fusion in alveolar rhabdomyosarcoma
by fluorescence in situ hybridization. Gene Cliromosome Cuncer 1995; 12:
18G192.
75. McManus AP, O’Reilly MJ, Pritchard-Jones K, ct ul. Interphase fluores-
cence in situ hybridization detection of t(2;13)(q35;q14)-a diagnostic tool
in minimally invasive biopsies. J Pathol (in press).
76. Downing JR, Khandekar A, Shurtleff SA, ef al. Multiplex RT-PCR assay
for the differential diagnosis of alveolar rhabdomyosarcoma and Ewing’s
sarcoma. A m J Pathol 1995; 1 4 6 626-634.
77. Douglass EC, Shapiro DN, Valentine M, Rowe ST. Alveolar rhabdo-
myosarcoma with the t(2; 13): cytogenetic findings and clinicopathologic
correlations. Med Pediatr Oncol 1993; 21: 83-87.
78. Reboul-Marty J, Quintana E, Moseri V, el 01. Prognostic factors of alveolar
rhabdomyosarcoma in childhood: an lnternational Society of Paediatric
Oncology study. Cancer 1991; 6 8 493498.
79. Parham DM, Shapiro DN, Downing JR, Webber BL, Douglass EC. Solid
alveolar rhabdomyosarcoma with the t(2;13). Report of two cases with
diagnostic implications. Am J Surg Path01 1994; 1 8 474478.
80. Pappos AS, Crist WM, Kuttesch J, er al. Tumor-cell DNA content predicts
outcome in children and adolescents with clinical groups 111 embryonal
rhabdomyosarcoma. J Clin Oncol 1993; 1 0 1901-1905.