ORIGINAL ARTICLE

An Immunohistochemical Algorithm to Facilitate

Diagnosis and Subtyping of Rhabdomyosarcoma: The
Children’s Oncology Group Experience
Raffaella A. Morotti, MD,* Kathleen K. Nicol, MD,w David M. Parham, MD,z Lisa A. Teot, MD,y
Julie Moore, HT,w John Hayes, PhD,w William Meyer, MD,z and Stephen J. Qualman, MDw

against intranuclear myogenic transcription factors are, at

Abstract: Immunohistochemistry remains the current ancillary present, the best available markers for confirming the diagnosis
method of choice in the pathologic evaluation of small blue of RMS. Their differential expression in reactive myogenic
round-cell tumors. In at least 20% of cases of rhabdomyosar- lesions, variability in ARMS versus ERMS, and absence in
coma (RMS), it is considered an essential factor in the final and/ undifferentiated sarcomas suggest new biologic questions to be
or differential diagnosis of the malignancy. Newer immuno- explored in future studies.
stains (antimyogenin, MyoD1) generated against intranuclear
myogenic transcription factors offer pathologists the best hope Key Words: rhabdomyosarcoma, immunohistochemistry,
for improving the sensitivity and specificity of RMS diagnosis. A myogenin, MyoD1, diagnosis
large series of RMS (956) were studied consecutively from the (Am J Surg Pathol 2006;30:962–968)
intergroup rhabdomyosarcoma study and children’s oncology
group files, along with multiple other malignant, benign or
reactive lesions. A panel of antibodies to muscle-related antigens
(myogenin, MyoD1, desmin, muscle-specific actin) was studied
using formalin-fixed, paraffin-embedded tissue, an avidin-biotin/
peroxidase complex immunohistochemical technique, antigen
R habdomyosarcomas (RMS) are the most frequent
malignant soft tissue tumors of childhood.18 The
spectrum of histological differentiation of this tumor
retrieval technique as appropriate, and automated immunos- varies, as better differentiated RMS have crossstriations
taining. Myogenin and MyoD1 were equally sensitive (positive or rhabdomyoblasts that allow for a confident morpho-
for 97% of RMS cases), with both also showing similar logic diagnosis without adjunct studies. In contrast, less
specificity (90% vs. 91% of cases) for the diagnosis of RMS. differentiated RMS resemble other small blue round-cell
Myogenin and MyoD1 staining were sometimes intact in areas tumors, and in at least 20% of RMS cases immuno-
of coagulative tumor necrosis, but negated by B5 fixation. histochemistry (IHC) is required either for definitive
Isolated, rare benign myogenin-positive nuclei were seen diagnosis or as an essential factor in the differential
infrequently in reactive lymph nodes. Specifically, both myo- diagnosis.29
genin and MyoD1 had significantly greater extent of expression The available immunohistochemical markers have
for alveolar RMS (ARMS) than embryonal RMS (ERMS) increased and evolved as the knowledge of skeletal
(both with P<0.001). Similarly, both myogenin (P = 0.001) myogenesis has progressed. The first skeletal muscle
and MyoD1 (P<0.001) had significantly higher expression for immunostains used for the diagnosis of RMS were
ARMS than RMS, not otherwise specified (NOS). They were predominantly against filamentous and oxidation-related
never expressed in undifferentiated sarcomas; however, reactive antigens. Actins, myosin, myoglobin, and desmin have
or regenerative myocytes did show expression. Immunostains proven useful in the diagnosis of RMS,8,21,23,31,32 even
though these markers lack absolute sensitivity and/or
specificity.22,27
From the *Department of Pathology, Mount Sinai School of Medicine,
New York; wDepartment of Laboratory Medicine, Children’s
Recent research into the regulation of myogenesis
Hospital, Columbus, OH; zDepartment of Pathology, University by transcription factors has opened a new era in the
of Arkansas for Medical Sciences and Arkansas Children’s Hospital, diagnosis of the more undifferentiated RMS. In 1992, a
Little Rock, AR; yDepartment of Pathology, Children’s Hospital of monoclonal antibody to the myogenic regulatory protein,
Pittsburgh, Pittsburgh, PA; and zDepartment of Hematology and MyoD1, was introduced for use on frozen tissue.10,16 It
Oncology, University of Oklahoma Health Sciences Center, Okla-
homa City, OK. proved to be an extremely sensitive and specific marker.
Supported in part by NIH/NCI Grants: CA 24507, CA72989 and CA Its use, however, on paraffin-embedded tissue historically
54021. gave less reliable results and thus many pathology
Reprints: Stephen J. Qualman, MD, Center for Childhood Cancer, laboratories do not routinely use it.39
WA5011, Columbus Children’s Research Institute, 700 Children’s
Dr Columbus, OH 43205 (e-mail: [email protected].
An immunohistochemical stain for myogenin,
edu, [email protected]). another intranuclear transcription factor that plays a role
Copyright r 2006 by Lippincott Williams & Wilkins early in myogenesis, has been more recently introduced.39

962 Am J Surg Pathol  Volume 30, Number 8, August 2006

Am J Surg Pathol  Volume 30, Number 8, August 2006 The Children’s Oncology Group Experience

To date, smaller retrospective reports have shown the tumors, benign tumors, and reactive lesions (Table 1).
utility of this immunohistochemical marker in the RMS were classified according to the international
diagnosis of RMS,4,6,9,14,20,39 and in helping to subtype classification of RMS (ICR).25 Assignment into a specific
RMS into alveolar and embryonal categories9,20; MyoD1 RMS subtype and scoring of immunostains was achieved
has not been previously studied as to its level of by consensus diagnosis of 3 reviewers (Drs Stephen
expression in RMS subtypes as compared with myogenin Qualman, David Parham and Lisa Teot), who indepen-
via IHC. dently evaluated all RMS cases.
Our study has semiquantitatively evaluated the Upon entering the study, all RMS and nonRMS
expression of myogenin, MyoD1, polyclonal-desmin, cases were stained with a panel of 4 striated muscle
and muscle-specific actin (MSA) in formalin-fixed, immunohistochemical stains which included two filament
paraffin-embedded sections, in a large series of soft tissue markers (muscle specific actin and desmin) and 2 intra-
sarcoma cases. In the present study we demonstrate, on a nuclear transcription factors (MyoD1 and myogenin).
large number of RMS (956) studied prospectively, that NonRMS cases were further characterized with other
both myogenin and MyoD1 are among the most sensitive immunostains depending upon the differential diagnosis
and specific for markers currently available for the considered.
diagnosis of RMS. Because previous molecular and Immunoperoxidase stains for cytoplasmic filaments
immunohistochemical studies have indicated differences and intranuclear transcription factors were performed on
in myogenin expression in the alveolar (ARMS) and paraffin-embedded, formalin-fixed tissue using standard
embryonal (ERMS) subtypes of rhabdomyosarco- avidin-biotin-peroxidase complex (ABC) technique,29
mas,9,19,33 our goal was to verify this observation and with monoclonal antiserum against actin (HHF 35,
its clinical applicability, as compared with MyoD1 MO635, DAKO Corp, Carpinteria, CA) at 1:100 dilution
expression, in a larger consecutive series of cases collected and polyclonal rabbit antiserum against desmin (DAKO
from the files of the intergroup rhabdomyosarcoma study Corp.) at 1:1000 dilution, without antigen retrieval
group (IRSG), now a clinical trial component of the technique. Monoclonal MyoD1 (DAKO Corp) (1:10
children’s oncology group (COG). dilution) and myogenin antisera (DAKO Corp.) (1:100
dilution) were also both utilized for immunoperoxidase
staining on formalin-fixed, paraffin-embedded tissue
MATERIALS AND METHODS using antigen retrieval technique. Four-micron thick
This study included 1052 consecutive cases collected sections were obtained and dried at 371C for 1 hour.
from IRSG files and consultation files from May, 1998 to Slides were deparaffinized and hydrated in distilled water.
July, 2004. This series included 956 rhabdomyosarcomas Endogenous peroxidase activity was blocked by immer-
(RMS), 31 undifferentiated sarcomas and 65 nonRMS sion in a 3% hydrogen peroxidase solution. After rinsing
cases, which consisted of a potpourri of malignant in distilled water selected slides were incubated in antigen

TABLE 1. Myogenic-related Markers in NonRMS Tumors

Diagnosis Myogenin Myod1 Desmin
Ewings/EOE/PNET (n = 12) Negative Negative Negative
Pleuropulmonary blastoma (n = 9) Positive (8) Positive (8) Positive (8)
Hematopoietic (n = 5)* Negative Negative Negative
Inflammatory myofibroblastic tumor (n = 6) Negative Negative Positive (6)
Rhabdoid tumor (n = 5) Negative Negative Positive (3)
Synovial sarcoma (n = 4) Negative Negative Positive (1)
Sarcoma NOS (n = 4) Negative Negative Negative
Undifferentiated embryonal sarcoma of liver (n = 4) Negative Negative Positive (2)
Epithelioid sarcoma (n = 3) Negative Negative Positive (2)
Fibrosarcoma (n = 2) Negative Negative Positive (1)
MFH (n = 2) Negative Negative Positive (1)
Angiomatoid fibrous histiocytoma (n = 1) Negative Negative Negative
Angiomyofibroblastoma (n = 1) Negative Negative Positive
Fibrous hamartoma of infancy (n = 1) Negative Negative Not Done
Mesenchymal hamartoma (n = 1) Negative Negative Positive (1)
Fibromatosis (n = 1) Negative Negative Negative
Epithelioid MPNST (n = 1)w Negative Negative Negative
Alveolar soft-part sarcoma (n = 1) Negative Negative Positive (1)
Langerhan cell histiocytosis (n = 1) Negative Negative Negative
Germ cell tumor with 10% ERMS (n = 1) Positive (1) Positive (1) Positive (1)
Sertoli-Leydig cell tumor (n = 1) Negative Negative Positive (1)
Osteosarcoma (n = 1) Negative Negative Negative
*‘‘Hematopoietic’’, Burkitt lymphoma, granulocytic sarcoma, lymphoblastic lymphoma, myelomonocytic leukemia, anaplastic large cell lymphoma.
wMPNST, malignant peripheral nerve sheath tumor.

r 2006 Lippincott Williams & Wilkins 963

Morotti et al Am J Surg Pathol  Volume 30, Number 8, August 2006

retrieval solution (Citra solution, Biogenex, San Ramon,

CA) and microwaved for 10 min. Primary antibody was
applied and kept at 41C overnight (preferably) or 1 h at
room temperature. The standard ABC technique29 was
then followed using an automated immunostainer
(DAKO Corp).
All immunostains were prospectively graded, in a
semiquantitative way, for all 4 muscle immunostains
relative to the percentage of immunopositive cells. The
scoring system adopted as published elsewhere29 was
as follows: 0 = absent staining, 1+ = minimal/focal
staining, 2+ = less than 10% of the cells positive,
3+ = 10 to 50% of the cells positive and 4+ = more
than 50% of the cells positive.

STATISTICAL ANALYSIS
Skeletal muscle immunostains (myogenin, MyoD1
and desmin) are described with sensitivity and specificity, FIGURE 1. Myogenin immunopositivity in benign regenera-
and tested for association across diagnoses with w2 test tive myocytes in posttreatment RMS.
analysis. Comparisons between diagnoses were made with
overall Kruskal-Wallis tests and paired tests were made
with Wilcoxon rank sums. Test statistics are considered
are always myogenin and MyoD1 immunonegative. All
significant at P<0.05.
31 cases of undifferentiated sarcomas were negative for
these 3 immunostains.
RESULTS Myogenin and MyoD1 were interpreted as positive
Our series included 1052 lesions of which 956 were only when positive immunostaining was detected in the
RMS, 31 undifferentiated sarcomas and 65 nonRMS nucleus. Differences in quality between the two stains
cases. RMS were classified as 508 ERMS, 416 ARMS and were sometimes noted. Myogenin was almost always
32 RMS-NOS (not otherwise specified); the latter due to clearly localized in the nucleus and the stain in the
limited diagnostic material. The nonRMS cases (Table 1) majority of cases was intense and well defined. With
included 12 peripheral primitive neuroectodermal tumor MyoD1, the stain was not always selectively localized in
(pPNET)/Ewing sarcomas, 9 pleuropulmonary blastomas the nucleus, but in occasional cases a cytoplasmic
(PPB), 5 rhabdoid tumors and lesser number of various background staining was also present; nevertheless, any
other lesions/tumors, including some having myofibro- nuclear staining was scored as positive, with or without
blastic characteristics (Table 1), such as inflammatory cytoplasmic staining. We have also noted a tendency of
myofibroblastic tumors (IMTs). the MyoD1 stain to become weaker with time. Sections
Results of the statistical analysis regarding sensiti- that were not stained within a few days after they were
vity and specificity of the 4 myogenic-related immuno- cut, or that were maintained at room temperature,
stains revealed that myogenin had a sensitivity of 97% showed weaker positivity and rare false negativity. Both
and a specificity of 90% (w2 = 726, P<0.001), MyoD1 MyoD1 and myogenin immunostains were routinely
had a sensitivity of 97% and a specificity of 91% negative with use of mercury-based fixatives (eg, B5
(w2 = 747, P<0.001), and p-desmin had a sensitivity of solution) for tissue fixation. This explains much of the 3%
99% and a specificity of 71% (w2 = 698, P<0.001). We immuno-negativity seen in some RMS which are evident
also used to include so-called ‘‘muscle-specific’’ actin in as to diagnosis by light microscopy alone. Surprisingly,
our immunostaining, published as having a 94% sensi- both MyoD1 and myogenin immunostains could survive
tivity29; in our hands, MSA had a sensitivity of 70%, and in areas of ischemic tumor necrosis (Fig. 2); thus such
has been subsequently dropped from our panel. positive immunostains should not be judged as a measure
Myogenin and MyoD1 were negative in 2% to 3% of tumor viability.
of ARMS/ERMS, and were focally positive in scattered In the different subtypes of RMS, the pattern of
rhabdomyoblasts in all but one of the PPB and in the staining varied (Table 2). Specifically, myogenin had
RMS component of a germ cell tumor. They were also significantly greater extent of expression than MyoD1 for
positive in reactive or regenerative myocytes underlying a ARMS (Wilcoxon signed rank =  2.8, P = 0.005) and
cutaneous ulcer bed as well as in such myocytes of a ERMS (Wilcoxon signed rank =  7.6, P<0.001).
posttreatment RMS in which residual tumor was not There was no difference between stains for RMS/NOS.
identified (Fig. 1). Desmin staining was negative in 1% of The overall Kruskall Wallis tests rejected the null
the RMS and reacted positively in the PPB, IMT and a hypothesis of equality of the tests to differentiate between
few of the other nonRMS cases. Desmin will clearly stain subtypes (myogenin P<0.001; MyoD1 P<0.001; Table
some myofibroblastic lesions (eg, IMT) routinely which 2). Specifically, both myogenin (P<0.001) and MyoD1

964 r 2006 Lippincott Williams & Wilkins

 Am J Surg Pathol  Volume 30, Number 8, August 2006 The Children’s Oncology Group Experience

FIGURE 2. Myogenin immunopositivity in necrotic RMS

nuclei.

(P<0.001) had significantly higher expression for ARMS

than ERMS. Similarly, both myogenin (P = 0.001) and
MyoD1 (P<0.001) had significantly higher expression
for ARMS than RMS/NOS. There were no significant
differences between ERMS and RMS/NOS for either
stain.
In the majority of ERMS the distribution of
myogenin positive cells (2 to 3+ or 10% to 50% of
nuclei) was patchy, with islands of intensely immuno-
positive cells alternating with fascicles of totally negative
cells (Fig. 3A). Conversely, ARMS showed a more
uniformly diffuse pattern of staining (Fig. 3B) (often a
4+ staining reaction or 50 to 100% of nuclei). Very rarely
we identified scattered, focal (1+ or less) benign
myogenin positive nuclei in lesions or tissues unrelated FIGURE 3. A, Distribution of myogenin staining (focal,
to RMS, including benign lymph nodes (Fig. 4A); these patchy) in ERMS. B, Diffuse, uniform myogenin positivity in
isolated benign nuclei were readily separable from ARMS.
the subtle nests of solid ARMS that could be found in
the sinusoids of lymph nodes containing subclinical
metastases (Fig. 4B). (embryonal RMS) or unfavorable (alveolar RMS,
undifferentiated sarcoma) histologic subtypes of diseases.
DISCUSSION Histopathologic subtype has repeatedly been shown to be
The precise pathological subtyping of rhabdomyo- a highly predictive independent prognostic factor36 both
sarcomas is important because important epidemiologi- in primary as well as recurrent RMS.26
cal, biologic, and treatment differences exist between Immunohistochemistry is the current method of
the subtypes. For example, in the IRSG III5 study, choice in the pathologic evaluation of a small blue round-
the enrollment onto specific treatment protocols was cell tumors (SBRCTs). Many of the immunohistochem-
predominantly based upon the diagnosis of favorable ical stains used for the diagnosis of RMS by pathology

TABLE 2. Distribution of Myogenin and MyoD1 Scores in RMS Subtypes (%)

Myogenin Score MyoD1 Score
Diagnosis 0 1+ 2+ 3+ 4+ 0 1+ 2+ 3+ 4+
ARMS 3 3 4 18 72 2 4 6 24 63
ERMS 3 3 13 38 42 3 4 19 58 16
RMS/NOS 11 19 15 7 48 14 7 18 43 18

r 2006 Lippincott Williams & Wilkins 965

Morotti et al Am J Surg Pathol  Volume 30, Number 8, August 2006

ARMS, however, do not show the classically described

translocations t(2;13) or t(1;13).2 In addition, the prac-
tical application of molecular studies, even in the light
of the faster results obtained by reverse transcriptase
polymerase chain reaction (RT-PCR), still are not the sole
test results on which to determine a treatment protocol.
In the present study, we have examined the
sensitivity and specificity of various immunohistochem-
ical markers for the diagnosis of rhabdomyosarcomas.
We found that the sensitivity and specificity of muscle
specific actin (MSA) is so low as to be unreliable for the
diagnosis and/or exclusion of RMS.
Desmin is currently a popular marker employed and
indeed it has a very good sensitivity, but lesser specificity
in the diagnosis of RMS; hence, when dealing with a
SBRCT, one cannot base a diagnosis of RMS solely on
desmin staining. It is well known,16 for example, that
intraabdominal desmoplastic small round cell tumor
(IADRCT) stains for desmin. In the pediatric population,
numerous other desmin-positive lesions, which mimic
RMS histologically, have also been described13,27,28,34,38;
these include not only IADRCT and IMT, but also
alveolar soft part sarcomas and angiomatoid fibrous
histiocytoma. The blastemal component of Wilms’ tumor
has also been shown to be positive for desmin in antigen-
retrieved sections.15 As noted in these examples, the
therapeutic consequences of a diagnosis based only on
desmin positivity can be significant. The absence of
desmin staining in RMS also may be problematic. In our
series, 1% of RMS failed to show desmin staining. In fact,
some authors have suggested that the more undifferen-
tiated RMSs are likely to show little or no reactivity for
this marker.12
WT1 antibody has recently been shown to have
variable positivity in pediatric small round blue cell
tumors; however the expression clearly is more limited to
differentiated RMS.3 Similarly, Goldsmith et a17 describe
the utilization of placental alkaline phosphatase (PLAP)
to confirm the presence of myogenic differentiation in a
variety of tumors.17 Confirmatory immunostaining of
differentiated RMS is helpful but not necessarily essential
to RMS diagnosis; the 20% of cases which require IHC
usually lack obvious myogenic differentiation.
FIGURE 4. A, Myogenin immunopositivity in benign nuclei In our series, MyoD1 and myogenin immunostains
(arrows) in lymph node parenchyma. B, Myogenin immuno- were the most useful for confirmation of the diagnosis of
positivity in sinusoidal nests of solid ARMS. RMS even without morphologic evidence of myogenic
differentiation. Myogenin and MyoD1 belong to the
MyoD family of myogenic helix-loop-helix (mHLHs)
laboratories are essentially markers for various cytopla- transcription factors.40 They coordinate the myogenic
smic filaments (predominantly desmin and frequently differentiation pathway from the determination of meso-
muscle-specific actin) or oxidative proteins (predomi- dermal precursors into myoblasts, the differentiation of
nantly myoglobin). However, these markers are either not myoblasts into myotubes, and finally the maturation of
specific to the myogenic lineage (desmin, actin) or are myotubes into skeletal myofibers.37 Myogenic cell lines
expressed infrequently and only in late stages of express each mHLH in a distinct temporal sequence
myogenesis (myoglobin). during differentiation. MyoD1 is expressed in myoblasts
Karyotypic and molecular studies have supported beforedifferentiation while myogenin has postdifferentia-
the morphologic differences of alveolar and embryonal tion functions.7,24,41 Immunostains for these transcription
rhabdomyosarcoma subtypes and may provide a more factors identify cells committed to myogenesis in their
objective subtyping of the tumor. A percentage of earliest phase.

966 r 2006 Lippincott Williams & Wilkins

Am J Surg Pathol  Volume 30, Number 8, August 2006 The Children’s Oncology Group Experience

We have shown that MyoD1 and myogenin have diffuse staining seen with MyoD1 and myogenin may be
similarly high sensitivities and specificities. In our helpful as an ancillary observation where the diagnosis of
experience, however, the MyoD1 immunostain may on ARMS is suggested by light microscopy. This finding,
occasion be more capricious than myogenin immuno- however, may offer insight into the relationship of these 2
stain. In paraffin-embedded, formalin-fixed sections, this subtypes with regard to the different phases of myogen-
stain is sometimes more difficult to read because of esis, and their different prognoses. The diffuse staining of
nonspecific cytoplasmic staining, which must be ignored; ARMS by myogenin and MyoD1 is also helpful in
only true intranuclear staining confirms a diagnosis of separating this subtype from RMS, NOS—the latter often
RMS.39 This may be one reason why many laboratories showing an ERMS-type histology but in a small enough
do not use this stain, even though it was introduced sample that subtyping is more difficult to confirm.
several years ago and numerous studies have shown the In conclusion, myogenin and MyoD1 are extremely
validity of this marker for the diagnosis of RMS.11,35 The sensitive and specific markers for the diagnosis of
percentage of MyoD1-positive RMS (97%) was higher in rhabdomyosarcomas and either could be used any time
our study compared with that of other groups where the this tumor is suspected. Positive myogenin and MyoD1
percentage of MyoD1 positivity was about 60% for the staining indicates the tumor to be derived from progeni-
embryonal and alveolar subtypes.1 This may be a tor cells committed toskeletal muscle differentiation or to
reflection of technical difficulties that were encountered contain such a component in heterogeneous tumors (eg,
with this stain by more than 1 pathology laboratory, the mixed germ cell tumor with RMS component, PPB). This
deterioration of the antigen in stored paraffin sections and observation has been previously made in Wilms’ tumors
the fact that our center has availed itself of refinements in containing a stromal component of fetal rhabdomyo-
immunohistochemical technique and interpretation (anti- blasts.14,39 Care should be taken in identifying reactive/
gen retrieval, automated staining, scoring of only clearly regenerative myocytes as rhabdomyoblasts; the clinical
identifiable intranuclear staining—with or without cyto- context of the lesion is usually a clue in this regard. We
plasmic positive staining). Certain practical caveats exist. currently perform the myogenin, MyoD1 and desmin
The identification of myogenin in isolated benign nuclei immunostains as a trio of markers, as any RMS is
of nonRMS tissues has been observed by others as by us virtually never negative with the three markers combined.
in reactive lymph nodes. The origin of these nuclei is The myogenin immunostain is more technically reliable
uncertain but they have been postulated to represent as a marker, given its lesser incidence of nonspecific
some sort of native nonneoplastic cell with myogenic cytoplasmic staining as compared with antiMyoD1, but
potential.4 Care must also be taken not to equate RMS rarely it will be completely negative in RMS in
immunopositivity for myogenin or MyoD1 with tumor the presence of strong intranuclear MyoD1 immuno-
viability (stain persists on ischemic tumor nuclei) or positivity.
immunonegativity as evidence of a nonRMS diagnosis in Undifferentiated sarcomas, unless demonstrated in
B5-fixed sections. the future to express new, even more primitive markers of
Both MyoD1 and myogenin stained the rhabdo- myogenesis than myogenin or MyoD1, most likely do not
myoblastic component in pleuropulmonary blastomas belong to this group of skeletal muscle tumors, despite
and germ cell tumors, as well as reactive/regenerative their current inclusion in the ICR classification for
myocytes in patients with other lesions (postchemothe- treatment purposes.30 Other unique molecular or anti-
rapy tissue, ulcer bed in eyelid). Although these results genic markers are needed to better define this subgroup of
have been considered as false positive cases in our tumors which show a poor prognostic outcome in current
analysis, it is evident that the histologic features and RMS clinical trials.
clinical data concerning these lesions should easily guide
the pathologist toward the correct diagnosis.
ACKNOWLEDGMENTS
In a recent study,9 it was shown that rhabdomyo-
sarcomas of the alveolar subtype show strong positivity The authors wish to thank Florinda Jaynes, HT for
for myogenin on frozen section. The authors suggested histotechnical work, and Mariko Suchi, MD, for contribu-
that the myogenin stain might be used as an adjunct for tion of Figure 4A.
distinguishing between alveolar and embryonal rhabdo-
myosarcoma. This is an important consideration, because REFERENCES
the differentiation of these 2 subtypes has major treat- 1. Alaggio R, D’Amore E, Dall’Igna P, et al. Pediatric rhabdomyo-
ment implications. Our data suggest that alveolar RMS sarcomas: an immunohistochemical study. Mod Pathol. 2000;
expresses greater staining for myogenin and MyoD1 13:202A.
2. Barr FG. Molecular genetics and pathogenesis of rhabdomyo-
(a 4+ reaction— 50% to 100% of nuclei in the majority sarcoma. J Pediatr Hematol Oncol. 1997;19:483–491.
of cases) than the embryonal subtype and with a different 3. Carpentieri DF, Nichols K, Chou PM, et al. The expression of WT1
histologic pattern. Even though our results achieved in the differentiation of rhabdomyosarcoma from other pediatric
statistical significance, the considerable interpretive over- small round blue cell tumors. Mod Pathol. 2002;15:1080–1086.
4. Cessna MH, Zhou H, Perkins SL, et al. Are myogenin and MyoD1
lap in degree of immunopositivity (Table 2) makes us expression specific for rhabdomyosarcoma? A study of 150 cases,
reluctant to use this as a primary tool to differentiate with emphasis on spindle cell mimics. Am J Surg Pathol. 2001;
embryonal from alveolar subtypes in daily practice. The 25:1150–1157.

r 2006 Lippincott Williams & Wilkins 967

Morotti et al Am J Surg Pathol  Volume 30, Number 8, August 2006

5. Crist W, Gehan EA, Ragab AH, et al. The third intergroup 24. Montarras D, Pinset C, Chelly J, et al. Expression of MyoD1
rhabdomyosarcoma study. J Clin Oncol. 1995;13:610–630. coincides with terminal differentiation in determined but inducible
6. Cui S, Hano H, Harada T, et al. Evaluation of new monoclonal anti- muscle cells. EMBO J. 1989;8:2203–2207.
MyoD1 and anti-myogenin antibodies for the diagnosis of 25. Newton WA Jr, Gehan EA, Webber BL, et al. Classification of
rhabdomyosarcoma. Pathol Int. 1999;49:62–68. rhabdomyosarcomas and related sarcomas. Pathologic aspects
7. Davis RL, Weintraub H, Lassar AB. Expression of a single and proposal for a new classification—an intergroup rhabdomyo-
transfected cDNA converts fibroblasts to myoblasts. Cell. 1987; sarcoma study. Cancer. 1995;76:1073–1085.
51:987–1000. 26. Pappo AS, Anderson JR, Crist WM, et al. Survival after relapse in
8. de Jong AS, van Kessel-van Vark M, Albus-Lutter CE, et al. children and adolescents with rhabdomyosarcoma: a report from the
Skeletal muscle actin as tumor marker in the diagnosis of intergroup rhabdomyosarcoma study group. J Clin Oncol. 1999;17:
rhabdomyosarcoma in childhood. Am J Surg Pathol. 1985;9: 3487–3493.
467–474. 27. Parham DM, Dias P, Kelly DR, et al. Desmin positivity in primitive
9. Dias P, Chen B, Dilday B, et al. Strong immunostaining for neuroectodermal tumors of childhood. Am J Surg Pathol. 1992;16:
myogenin in rhabdomyosarcoma is significantly associated with 483–492.
tumors of the alveolar subclass. Am J Pathol. 2000;156:399–408. 28. Parham DM, Webber B, Holt H, et al. Immunohistochemical study
10. Dias P, Parham DM, Shapiro DN, et al. Monoclonal antibodies to of childhood rhabdomyosarcomas and related neoplasms. Results of
the myogenic regulatory protein MyoD1: epitope mapping and an intergroup rhabdomyosarcoma study project. Cancer. 1991;67:
diagnostic utility. Cancer Res. 1992;52:6431–6439. 3072–3080.
11. Dias P, Parham DM, Shapiro DN, et al. Myogenic regulatory 29. Qualman SJ, Coffin CM, Newton WA, et al. Intergroup rhabdo-
protein (MyoD1) expression in childhood solid tumors: diagnostic myosarcoma study: update for pathologists. Pediatr Dev Pathol.
utility in rhabdomyosarcoma. Am J Pathol. 1990;137:1283–1291. 1998;1:550–561.
12. Eusebi V, Ceccarelli C, Gorza L, et al. Immunocytochemistry of 30. Raney RB, Anderson JR, Barr FG, et al. Rhabdomyosarcoma and
rhabdomyosarcoma. The use of four different markers. Am J Surg undifferentiated sarcoma in the first two decades of life: a selective
Pathol. 1986;10:293–299. review of intergroup rhabdomyosarcoma study group experience
13. Fanburg-Smith JC, Miettinen M. Angiomatoid ‘‘malignant’’ fibrous and rationale for intergroup rhabdomyosarcoma study V. J Pediatr
histiocytoma: a clinicopathologic study of 158 cases and further Hematol Oncol. 2001;23:215–220.
exploration of the myoid phenotype. Hum Pathol. 1999;30: 31. Schmidt RA, Cone R, Haas JE, et al. Diagnosis of rhabdomyo-
1336–1343. sarcomas with HHF35, a monoclonal antibody directed against
14. Folpe AL. MyoD1 and myogenin expression in human neoplasia: a muscle actins. Am J Pathol. 1988;131:19–28.
review and update. Adv Anat Pathol. 2002;9:198–203. 32. Scupham R, Gilbert EF, Wilde J, et al. Immunohistochemical
15. Folpe AL, Patterson K, Gown AM. Antibodies to desmin identify studies of rhabdomyosarcoma. Arch Pathol Lab Med. 1986;110:
the blastemal component of nephroblastoma. Mod Pathol. 1997; 818–821.
10:895–900. 33. Tonin PN, Scrable H, Shimada H, et al. Muscle-specific gene
16. Gerald WL, Miller HK, Battifora H, et al. Intra-abdominal expression in rhabdomyosarcomas and stages of human fetal
desmoplastic small round-cell tumor. Report of 19 cases of a skeletal muscle development. Cancer Res. 1991;51:5100–5106.
distinctive type of high-grade polyphenotypic malignancy affecting 34. Truong LD, Rangdaeng S, Cagle P, et al. The diagnostic. utility of
young individuals. Am J Surg Pathol. 1991;15:499–513. desmin. A study of 584 cases and review of the literature. Am J Clin
17. Goldsmith JD, Pawel B, Goldblum JR, et al. Detection and Pathol. 1990;93:305–314.
diagnostic utilization of placental alkaline phosphatase in muscular 35. Tsokos M, Dias P, Jefferson J, et al. Detection of the MyoD1 gene
tissue and tumors with myogenic differentiation. Am J Surg Pathol. product in paraffin section of pediatric tumors. Mod Pathol.
2002;26:1627–1633. 1994;7:148.
18. Gurney JG, Severson RK, Davis S, et al. Incidence of cancer in 36. Tsokos M, Webber BL, Parham DM, et al. Rhabdomyosarcoma.
children in the United States. Sex-, race-, and 1-year age-specific A new classification scheme related to prognosis. Arch Pathol Lab
rates by histologic type. Cancer. 1995;75:2186–2195. Med. 1992;116:847–855.
19. Hostein I, Andraud-Fregeville M, Guillou L, et al. Rhabdomyo- 37. Venuti JM, Cserjesi P. Molecular embryology of skeletal myogen-
sarcoma: value of myogenin expression analysis and molecular esis. Curr Top Dev Biol. 1996;34:169–206.
testing in diagnosing the alveolar subtype: an analysis of 109 38. Wang NP, Bacchi CE, Jiang JJ, et al. Does alveolar soft-part
paraffin-embedded specimens. Cancer. 2004;101:2817–2824. sarcoma exhibit skeletal muscle differentiation? An immunocyto-
20. Kumar S, Perlman E, Harris CA, et al. Myogenin is a specific chemical and biochemical study of myogenic regulatory protein
marker for rhabdomyosarcoma: an immunohistochemical study in expression. Mod Pathol. 1996;9:496–506.
paraffin-embedded tissues. Mod Pathol. 2000;13:988–993. 39. Wang NP, Marx J, McNutt MA, et al. Expression of myogenic
21. Leader M, Patel J, Collins M, et al. Myoglobin: an evaluation of its regulatory proteins (myogenin and MyoD1) in small blue round cell
role as a marker of rhabdomyosarcomas. Br J Cancer. 1989; tumors of childhood. Am J Pathol. 1995;147:1799–1810.
59:106–109. 40. Wright WE, Dac-Korytko I, Farmer K. Monoclonal antimyogenin
22. Miettinen M. Antibody specific to muscle actins in the diagnosis and antibodies define epitopes outside the bHLH domain where binding
classification of soft tissue tumors. Am J Pathol. 1988;130:205–215. interferes with protein-protein and protein-DNA interactions. Dev
23. Molenaar WM, Oosterhuis JW, Oosterhuis AM, et al. Mesenchymal Genet. 1996;19:131–138.
and muscle-specific intermediate filaments (vimentin and desmin) in 41. Wright WE, Sassoon DA, Lin VK. Myogenin, a factor regulating
relation to differentiation in childhood rhabdomyosarcomas. Hum myogenesis, has a domain homologous to MyoD. Cell. 1989;56:
Pathol. 1985;16:838–843. 607–617.

968 r 2006 Lippincott Williams & Wilkins