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Short Communications |
From the Department of Cell and Molecular
Biology,*
Imgenex Corporation, San Diego, California; the
Departments of Pathology and Laboratory Medicine and
Pathology,
University of Arkansas for Medical
Sciences, Little Rock, Arkansas; the Department of
Pediatrics,
Kyoto Prefectural University of
Medicine, Kyoto, Japan; the Department of
Molecular Pharmacology,§
St. Jude Children’s
Research Hospital, Memphis, Tennessee; and the
Pediatric Division,¶
Cooperative Human Tissue
Network, Children’s Hospital, Columbus, Ohio
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The conventionally accepted histological classification scheme of Horn and Enterline2 recognizes embryonal, botryoid, alveolar, and pleomorphic tumors as distinct subtypes of rhabdomyosarcomas. Recent studies have shown that additional subgroups may be identified based on prognostic data.3 The need to accurately distinguish between the subtypes of rhabdomyosarcomas is becoming increasingly clear as we realize that distinct entities exhibit different clinicopathological behaviors and resulting prognosis.3-12 Alveolar rhabdomyosarcomas are particularly aggressive because of their capacity for invasive growth and metastasis in the early stages of the disease.3,6,11,12 However, it is sometimes difficult to distinguish between alveolar and embryonal rhabdomyosarcomas by histology alone.5
Cytogenetic data may be used to distinguish between the two subtypes, as embryonal rhabdomyosarcomas show deletion of chromosome 11p13-15 and alveolar rhabdomyosarcomas show reciprocal translocations involving chromosomes 116,17 or 218-20 with chromosome 13. However, cytogenetic analysis is technically difficult, and fewer than 50% of tumors can successfully be karyotyped.21 Although polymerase chain reaction (PCR) may be used for diagnosing alveolar rhabdomyosarcomas, using primers that amplify the Pax3 or Pax 7-Forkhead (FKHR) chimeric genes involved in the chromosome translocation, this technique is not widely available in clinical laboratories, and furthermore it requires the destruction of tissue. Apart from histology and cytogenetic characteristics, there are currently no definitive protein markers that can accurately distinguish between the subclasses of rhabdomyosarcomas. Therefore it is desirable to identify new markers that may be used to distinguish between the different subtypes of rhabdomyosarcomas. Such markers could be clinically useful diagnostic tools and make possible accurate comparisons of different therapeutic protocols.
The myogenic determination (MyoD) gene, the human homolog of which is known as myf3, was first identified by virtue of its ability to convert nonmuscle cells into muscle cells.22 Subsequent to the identification of MyoD, three other myogenic regulatory genes, myf5, myogenin (the human homolog of which is myf4), and MRF4 or herculin (the human homolog of which is myf6),23-25 were identified. The products encoded by these genes share sequence homology with MyoD, induce myogenic differentiation, and constitute the family of myogenic regulatory factors.26-28 Antibodies to MyoD can be used to diagnose rhabdomyosarcomas from other pediatric and adult tumors.29-32 In the present study we provide preliminary evidence showing that the intensity and frequency of immunostaining of tumor cells for myogenin can be used to distinguish alveolar rhabdomyosarcomas from embryonal rhabdomyosarcomas.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 26 rhabdomyosarcomas were obtained from collaborating institutions of the Intergroup Rhabdomyosarcoma Study (IRS)33 or the tumor bank of St. Jude Children’s Research Hospital. All tissues were snap frozen in liquid nitrogen and stored at -70°C. Clinical data for all tissues were obtained from the respective institutions. For each tumor, the diagnosis of rhabdomyosarcoma was confirmed from available clinical and histopathological data that also included confirmed positive staining for other myogenic markers such as desmin and MyoD.30 Analysis for PAX 3/7-FKHR was performed by polymerase chain reaction/reverse transcriptase-polymerase chain reaction (PCR/RT-PCR). The histological subclassification of all tumors used in the study was obtained from IRS Pathology review.33
To confirm that there were differences in expression of myogenin between embryonal and alveolar rhabdomyosarcomas, a subgroup of 12 additional rhabdomyosarcomas obtained from the Cooperative Human Tissue Network (CHTN, Columbus, Ohio) were subsequently analyzed for quantitative differences in expression of myogenin by Western blot analysis. These tumors were also examined by immunohistochemistry to confirm data from Western blotting.
Antibodies
The monoclonal antibody to myogenin (Imgenex, San Diego, CA) was
raised against glutathione S-transferase (GST)-myogenin
fusion protein. The antibody was found to react specifically with a
peptide sequence that corresponds to amino acid residues 144–158 of
rat myogenin protein.34
This region is specific to
myogenin and falls distal to the second helix of the HLH domain.
Although the monoclonal antibody to myogenin was raised against rat
myogenin fusion protein, it cross-reacts with human (myf4) and mouse
myogenin, and its specificity for myogenin was demonstrated using a
panel of small round cell tumor cell lines, including an alveolar
rhabdomyosarcoma cell line (Figure 1)
. A
monoclonal antibody to desmin (clone D33; Dako, Carpenteria, CA) was
used as an isotype-matched positive control antibody, and a monoclonal
antibody to leukocyte common antigen (CD45) (clone 2B11; Dako) was used
as an isotype-matched negative control antibody.
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Cell Lines
To confirm the specificity of the monoclonal antibody for myogenin, cell lysates were prepared from a panel of small round tumor cell lines and tested by Western blotting. The cell lines included Rh30 (alveolar rhabdomyosarcoma), PFSK-1A (primitive neuroectodermal tumor), EB2 (lymphoma), SKNSH (neuroblastoma), and SJSA-1 (Ewing’s sarcoma). All cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD). For each cell line, cell lysates were prepared by homogenizing in a lysis buffer (10 mmol/L Tris (pH 7.5), 130 mmol/L NaCl, 1% Triton X-100, 10 mmol/L NaF, 10 mmol/L NaPi (pH 7.5), 10 mmol/L NaPPi pH 7.5). The protein concentration of each lysate was determined and used to load an equal amount of protein per well (10 µg/well) of a sodium dodecyl sulfate/polyacrylamide gel. The proteins were resolved through the gel, transferred to a nylon membrane, and tested for immunoreactivity with the antimyogenin antibody as initially described by Towbin et al.35 An isotype-matched IgG1 antibody was used as a negative control. For Western blotting, purified antibodies were diluted to 2 µg/ml in 5% nonfat powdered milk in Tris-buffered saline Tween (TBST) blocking buffer, whereas antibody-containing hybridoma culture supernatants were used at 1:3 dilution in the same blocking buffer. Excess unbound primary antibody was washed off, and the membrane was incubated with horseradish peroxidase-conjugated anti-mouse Ig (Jackson Immuno Research Laboratories, West Grove, PA). Antibody binding was detected using the Super Signal chemiluminescent substrate (Pierce, Rockford, IL) and photographic films (Eastman Kodak Company, Rochester, NY).
Tumor Tissue
Tumor lysates were obtained from an additional 12 rhabdomyosarcoma specimens, using lysis buffer as described above. For each tumor lysate, an equal amount of protein was loaded per well (10 µg/well) and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were then transferred to a nylon membrane and tested for immunoreactivity with 2 µg/ml anti-myogenin antibody as described above. Antibody reactivity with each tumor lysate was analyzed by densitometry, using an Alpha Innotech image analysis system (Alpha Innotech Corporation Corporation, San Leandro, CA).
Immunohistochemical Staining
Immunohistochemical staining for myogenin, desmin, and CD45 was performed by the avidin-biotin-complex (ABC) peroxidase method36 with a Vectastain kit (Vector Laboratories, Burlingame, CA). Briefly, 5-µm cryosections of tumor tissue on slides were fixed in 1% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes, permeabilized in 0.25% Triton X-100 in PBS for 20 minutes, and incubated with 1% bovine serum albumin in PBS for 30 minutes to block for nonspecific staining. The rest of the staining method was performed as previously described.30 All tissues were incubated with the primary antibody overnight at 4°C. For each tumor, serial sections were used for staining with myogenin, desmin, and CD45. The antibody to myogenin was used at an approximate antibody concentration of 5 µg/ml as a culture supernatant. The antibodies to desmin and CD45 were used in respective PBS dilutions of 1:400 and 1:100 according to the manufacturer’s instructions.
Scoring and Analysis of the Staining Data
For each tumor specimen, the antibody-stained sections were
examined independently by P. D., D. P., B. D., H.
H., S. Q., and J. T. Positive staining was scored when there
was discrete staining of cell nuclei that were not in close proximity
to the edge of the section, tissue folds, or areas of necrosis.
Staining was scored as follows: -, all tumor cells negative; +,
approximately 1–10% tumor cells moderately positive (see Figure 2
, top panels); ++, approximately
10–50% tumor cells moderately positive; +++, >50–100% tumor cells
strongly positive (see Figure 2
, bottom panels).
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and Figure 2
; compare
upper (+) panels with lower (+++) panels). The two myogenin-stained
tumor groups (groups A and B) were then compared with histological
classification, using Fisher’s exact test.
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Analysis for the PAX 3/7-FKHR fusion RT-PCR or PCR product in rhabdomyosarcoma specimens was performed on both frozen and formalin-fixed (cases 11, 18, 19, and 22) or alcohol-fixed (case 17) paraffin-embedded tissue. Paraffin-embedded tissue was first cut into 10-µm sections (10–20 sections each, depending on tissue volume) and deparaffinized with 10 changes of xylene for 30 minutes each at room temperature. The tissue was then rehydrated in a decreasing concentration of ethanol, one change each in 100%, 80%, 50%, and finally RNase-free water. Total RNA was extracted from frozen or deparaffinized tissue, using the Purescript RNA Extraction Kit according to the manufacturer’s instructions (Gentra Systems, Minneapolis, MN). For detection of the Pax3-FKHR or Pax7-FKHR fusion mRNA transcripts, the first strand of cDNA was synthesized by reverse transcription (AMV reverse transcriptase; Promega, Madison, WI) of RNA from the tumor tissue in the presence of deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP). PCR was performed as described previously,37 with the same FKHR reverse primer as used in the reverse transcription and a consensus Pax3/7 primer that recognizes both Pax3 and Pax7. This primer pair amplifies a 219-bp fragment for Pax3-FKHR and a 206-bp fragment for Pax7-FKHR.
A control amplification reaction was performed for each RNA sample, using the FKHR reverse primer and the FKHR forward primer to amplify the normal FKHR transcript. The cycling parameters for both PCR reactions were as previously described.37 The PCR product was resolved through a 10% native polyacrylamide gel and visualized with ethidium bromide under UV light. To confirm the nature of the fusion genes, the samples that were positive for translocations were then amplified separately, using the same FKHR reverse primer and primers specific for Pax3 or for Pax7. For the four paraffin-embedded tissues, a second-round PCR was performed using a set of seminested primers, including the FKHR forward, Pax3, and Pax7 specific primers together with a nested primer, FKHR C (5' TCT GCA CAC GAA TGA ACT 3'). The amplification and gel analysis procedures were as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-myogenin monoclonal antibody reacted with a single band
with an approximate size of 34 kd, which corresponds to the molecular
size of myogenin in the Rh30 alveolar rhabdomyosarcoma cell lysate
(Figure 1)
. All other small round cell tumor cell lines, including
primitive neuroectodermal tumor (PFSK-1A), lymphoma (EB2),
neuroblastoma (SKNSH), and Ewing’s sarcoma (SJSA-1), were negative
with the antibody. The Rh30 alveolar rhabdomyosarcoma cell line had
previously been shown to express myogenin transcripts by Northern
blotting.30
Immunohistochemical Staining of Tumor Specimens
Twenty-two of the 26 rhabdomyosarcomas were positive for myogenin.
Various patterns of staining for myogenin were observed (Table 1)
. Four
of the 17 embryonal rhabdomyosarcomas were negative for myogenin. Six
of the 17 tumors belonging to this subclass had occasional weakly
positive tumor cells, and five of the 17 embryonal rhabdomyosarcomas
had approximately 10–50% myogenin-positive tumor cells. The top
panels of Figure 2
show representative staining of most embryonal
rhabdomyosarcoma. Only two of the 17 embryonal rhabdomyosarcomas (cases
16 and 17) showed strong staining for myogenin in most of the tumor
cells. One of these two embryonal tumors (case 17) was subsequently
found to be positive for Pax3/FKHR (Table 1)
, whereas RT-PCR was not
performed on the second case, as a tissue specimen was not available.
In contrast, all alveolar rhabdomyosarcomas showed strong staining
in most of the tumor cells. Almost every alveolar rhabdomyosarcoma had
in excess of 70% strongly positive cells. The bottom panels of Figure 2
show representative staining of alveolar rhabdomyosarcomas. In
general, it appeared as if all tumor cells in alveolar
rhabdomyosarcomas were clonally derived from a myogenin-positive
progenitor cell. In contrast, a smaller proportion of cells in
embryonal rhabdomyosarcomas were positive for myogenin against a
background of distinctly negative cells. In all myogenin-positive
tumors distinct staining of cell nuclei was observed. There appeared to
be no strict correlation with staining for myogenin and the
morphological differentiation of the tumor cells. Thus strong staining
of a high proportion of tumor cells was observed both in cases with
unequivocal alveolar histology and in solid variant alveolar
rhabdomyosarcomas (tumors with dense sheets of undifferentiated
monomorphic tumor cells). However, weak to strong nuclear staining of
tumor cells that had acquired a higher status of differentiation was
also observed in embryonal rhabdomyosarcomas. Interestingly, one of the
two embryonal rhabdomyosarcomas that strongly stained for myogenin
appeared monomorphous, with large rounded nuclei and scanty cytoplasm
resembling the solid variant forms of alveolar rhabdomyosarcoma.
The staining results for the group A and B tumors are summarized on
Table 2
. Nine of the 11 tumors that
strongly stained for myogenin were alveolar rhabdomyosarcomas and only
two were embryonal rhabdomyosarcomas. One of the two embryonal
rhabdomyosarcomas (case 17) was characterized as positive for
Pax3-FKHR, indicating retrospectively that it was an alveolar
rhabdomyosarcoma. When the tumors were examined for correlation of
staining (group A versus group B) with tumor
subclassification (embryonal versus alveolar) by Fisher’s
exact test, there was a highly significant difference
(P = <0.0001) in the staining for myogenin
between embryonal and alveolar rhabdomyosarcomas.
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RNA samples were available from 20 of 26 tumors to examine for the Pax3–7/FKHR chimeric transcripts characteristic of alveolar rhabdomyosarcomas. Only one of the 13 embryonal rhabdomyosarcomas (case 17) examined was positive for Pax-3/FKHR transcripts. In contrast, six of seven alveolar rhabdomyosarcomas were positive for the Pax3–7/FKHR transcripts. Of these six alveolar tumors, five were positive for Pax3/FKHR and one (case 23) was positive for Pax7/FKHR transcripts. The one tumor that was negative for the Pax3–7/FKHR chimeric transcripts (case 26) appeared to be alveolar rhabdomyosarcoma by histology. This tumor was also strongly positive for myogenin.
Analysis of an Additional Subset of Rhabdomyosarcomas for Quantitative Differences in Expression of Myogenin by Western Blot Analysis and Correlation with Immunohistochemical Staining Data
Western blot analysis was carried out on 12 additional
rhabdomyosarcomas to test whether the difference in expression of
myogenin between embryonal and alveolar rhabdomyosarcoma observed by
immunohistochemistry could be shown quantitatively.
Molecular/cytogenetic data were unavailable for these 12 tumor cases.
There was a good correlation (P = 0.01, Pearson
Correlation) between densitometric readings for the Western blot and
the levels of staining for myogenin by immunohistochemistry (Figure 3
and Table 3
), although the latter technique was
more sensitive. Thus some tumors that were weakly positive by
immunohistochemistry were completely negative by Western blotting (see
Figure 3
, lanes 3, 4, and 6, and Table 3
, cases 3, 4, and 6). High
levels of myogenin were detected in four of six alveolar
rhabdomyosarcomas (Figure 3
, lanes 8, 10, 11, and 12, and Table 3
,
cases 8, 10, 11, and 12). Low levels of myogenin were detected in one
of the remaining two tumors (Figure 3
, lane 9), whereas none was
detected in the other (Figure 3
, lane 7). By immunohistochemistry, the
myogenin staining pattern of the weakly positive tumor (Figure 3
, lane
9, corresponding to Table 3
, case 9) resembled that typically observed
for embryonal rhabdomyosarcomas (figure not shown). Interestingly, the
alveolar rhabdomyosarcoma that was negative for myogenin by both
immunohistochemistry and Western blotting (Figure 3
, lane 7, and Table 3
, case 7) was also found to be negative for desmin by
immunohistochemistry. Desmin is an intermediate filament protein that
is often expressed in rhabdomyosarcomas.
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, lane 1, and Table 3
, case 1), which showed high levels of
myogenin, all other embryonal rhabdomyosarcomas either were negative
(Figure 3
, lanes 3, 4, and 6) or showed low levels of myogenin (Figure 3
, lanes 2 and 5). This was also reflected in the immunohistochemical
staining data, although, as mentioned above, tumors with a low
proportion and intensity of myogenin-positive cells were found to be
negative by Western blotting. Although the posttreatment embryonal
tumor was strongly positive for myogenin by Western blotting, the
staining pattern of this tumor was similar to that observed for
embryonal rhabdomyosarcomas in that, even though there was a higher
proportion of strongly myogenin-positive cells, there was also a
significant proportion of cells that were distinctly negative for
myogenin.
It was unfortunate that molecular/cytogenetic data, particularly for
the two questionable alveolar rhabdomyosarcomas (Figure 3
, lanes 7
and 9), were not available. Despite the unexpectedly low levels of
myogenin in these two alveolar rhabdomyosarcomas, on average,
densitometric analysis of the blot revealed that there was at least
threefold more myogenin in the alveolar rhabdomyosarcomas than in the
embryonal rhabdomyosarcomas. We anticipate that this difference may be
even larger and a stronger correlation may be observed if a larger
sample size of molecularly characterized tumors is used to validate the
present observation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To confirm the observation made by immunohistochemical staining, an additional 12 tumors were quantitatively examined for differences in expression of myogenin by Western blotting. There was a strong correlation in the results from the two techniques, in that higher levels of myogenin were detected in alveolar rhabdomyosarcomas as opposed to embryonal rhabdomyosarcomas. Densitometric analysis of the blot revealed that there was on average at least threefold more myogenin in the alveolar rhabdomyosarcomas than in the embryonal tumors. Although Western blot analysis was the more quantitative of the two techniques, it was less sensitive for detecting weak expression of myogenin in tumors. Furthermore, the immunohistochemical staining patterns for myogenin could clearly be used to distinguish between embryonal and alveolar rhabdomyosarcomas.
From the latter series, there were two alveolar rhabdomyosarcomas with
unexpectedly low levels of myogenin; one that was negative and one was
weakly positive for myogenin. Unfortunately, there are no cytogenetic
or molecular data to confirm the diagnosis of these tumors. One of
these two tumors was negative for desmin and myogenin and yet appeared
to be an alveolar rhabdomyosarcoma by histology. If tissue preservation
was not an issue, then, based on our immunostaining data, it is
possible that this tumor is not an alveolar rhabdomyosarcoma. The one
embryonal rhabdomyosarcoma that showed strong myogenin expression was a
posttreatment tumor. It is not surprising that this tumor was strongly
positive for myogenin, as induction of differentiation of tumor cells
by therapeutic agents is well documented. A combination of chemotherapy
and radiation typically induces a strong cytodifferentiation response
in rhabdomyosarcomas.38-40
It has also been shown that
the induction of differentiation by agents such as retinoic acid is
associated with activation and increases expression of the myogenin
gene.41
These observations thus suggest that the strong
myogenin expression in our single embryonal rhabdomyosarcoma case was a
result of the tumor therapy and not an intrinsic component of this
tumor. Despite the high levels of myogenin in this tumor, which is a
feature we suggest characterizes alveolar rhabdomyosarcomas, by
immunohistochemistry this tumor could clearly be distinguished as an
embryonal rhabdomyosarcoma. Essentially, the pattern of staining was
typical of embryonal tumors in that although there were areas of strong
focal staining for myogenin, a fairly large fraction of the tumor cells
were negative for myogenin, unlike the staining pattern observed in
alveolar rhabdomyosarcomas, where most of the cells were positive. In
this smaller subset of tumors, the difference in staining between
embryonal and alveolar rhabdomyosarcomas failed to reach statistical
significance, although there was a trend toward alveolar
rhabdomyosarcomas association with strong myogenin staining (Table 3)
.
A study using a larger number of molecularly characterized
rhabdomyosarcoma specimens will help to confirm our observations.
Other groups42-45 have previously examined the expression of myogenin in rhabdomyosarcomas. Tonin et al42 noted that all eight of their alveolar rhabdomyosarcomas expressed myogenin, whereas only five of eight embryonal rhabdomyosarcomas expressed myogenin transcripts. In the study of Clark et al43 transcripts of myogenin were detected in 10 of 15 embryonal rhabdomyosarcomas, whereas all three alveolar rhabdomyosarcomas plus one alveolar/embryonal mixed tumor expressed myogenin. Because expression was analyzed by Northern blotting in both studies42,43 it was not possible to tell the level of expression of myogenin per cell and whether all cells expressed myogenin. Using antibodies to MyoD and myogenin, Wang et al45 examined expression at the protein level. They noted that 22 of their 25 embryonal rhabdomyosarcomas were positive for myogenin, whereas four of four alveolar rhabdomyosarcomas were positive for myogenin. Unfortunately, these authors did not compare the intensity and frequency of staining between the subgroups of rhabdomyosarcomas. None of the above studies42,43,45 noted a correlation between the expression of the myogenic regulatory proteins and histological subtypes of rhabdomyosarcomas. Nevertheless it is interesting that in each of these studies some of the embryonal rhabdomyosarcomas were negative for myogenin, whereas all of the alveolar rhabdomyosarcomas were positive for myogenin. In the study of Hosoi et al44 myogenin expression was examined in two sublines derived from a human rhabdomyosarcoma tumor cell line. Interestingly, stronger expression for myogenin was observed in the subline that appeared to be more differentiated, which is consistent with the role of the protein in myogenic differentiation.
In normal myogenesis, differentiation is an orderly process involving epigenetic regulation of gene expression in the different stages of the myogenic differentiation pathway. Each stage is associated with down-regulation of specific sets of genes and simultaneous transcriptional up-regulation of others. The members of the myogenic bHLH family exhibit different spatiotemporal patterns of expression during embryogenesis.46-48 Each member may act at a different stage of the myogenic pathway. Through gene inactivation studies in mice it has been shown that whereas either myf 5 or MyoD is necessary for earlier stages of myogenesis,49 myogenin is obligatory for maturation of muscle.50 It is anticipated that if a tumor were to occur as a result of a block anywhere in this myogenic differentiation pathway, then the genes expressed by the tumor cells would reflect the stage at which the block in differentiation had occurred. In this regard, we propose that embryonal rhabdomyosarcomas are associated with a block in differentiation before the full expression of myogenin. The relatively small proportion of myogenin-positive cells in these tumors may reflect phenotypic variation due to the environment cues. It is noteworthy that strong staining for myogenin is associated with alveolar rhabdomyosarcomas, because these tumors usually appear to be undifferentiated by morphology. The fact that most of the tumor cells are myogenin positive would seem to suggest that alveolar tumors are clonally derived from myogenin-positive progenitor cells. We propose that either alveolar rhabdomyosarcomas result from a block in differentiation postexpression of myogenin, or that the pathological mechanisms involved in these neoplasms also activate strong myogenin expression. The fact that Pax3/7-FKHR transcripts were detected in six of seven alveolar rhabdomyosarcomas that were strongly positive for myogenin would support the latter notion. However, the one tumor that was negative for Pax3/7-FKHR transcripts indicates that other mechanisms may activate strong expression of myogenin in alveolar rhabdomyosarcoma.
Because there are significant differences in survival between patients with alveolar and embryonal rhabdomyosarcomas,11,12 our data suggest that strong expression of myogenin in untreated rhabdomyosarcomas is a marker of poor prognosis. It is perplexing that myogenin is strongly expressed in tumors associated with poor prognosis, because, normally, both MyoD51 and myogenin induce the expression of p21, a tumor suppressor gene, the product of which functions as an inhibitor of cyclin-dependent kinases and causes cells to exit the cell cycle. Because differentiation occurs when the forces that drive proliferation are minimized, it would be anticipated that strong expression of myogenin in untreated rhabdomyosarcomas would result in less aggressive and more differentiated tumors. However, alveolar rhabdomyosarcomas are normally viewed as tumors with an aggressive clinical behavior and poor prognosis.
It is now well established that translocation involving chromosomes 1 or 2 with chromosome 13 is characteristic of alveolar rhabdomyosarcomas. As mentioned above, PCR or RT-PCR for the translocation-associated Pax3/7-FKHR chimeric gene/transcript can be used to diagnose alveolar rhabdomyosarcomas. However, this technique is not widely available in clinical laboratories, whereas immunohistochemistry is widely used for diagnosis. Furthermore, morphological details may be missed by PCR/RT-PCR. From our study we observe that most alveolar rhabdomyosarcomas that strongly stained for myogenin were also positive for Pax3–7/FKHR by PCR/RT-PCR. In fact, one of two embryonal rhabdomyosarcomas that stained strongly for myogenin was also retrospectively found to be positive for Pax3/FKHR transcripts. This indicates that there is a strong association between the translocation-associated Pax3–7/FKHR fusion gene/transcripts and myogenin expression. Therefore we propose that staining for myogenin will be a useful and simple diagnostic test for distinguishing alveolar from embryonal rhabdomyosarcomas. The role of the chimeric gene/transcripts is still not fully understood. The resulting Pax7- or Pax3/FKHR chimeric proteins are potentially oncogenic.52,53 Epstein et al (1995)52 showed that the ectopic expression of both Pax3 and Pax3/FKHR was able to inhibit myogenic differentiation of MyoD-expressing 10T1/2 fibroblast cells when these cells were exposed to media with low levels of serum. Interestingly, they observe that Pax-3/FKHR was a more potent inhibitor of myogenesis than Pax-3 alone. In contrast, they showed that under the same conditions, Pax-3 and Pax-3/FKHR were unable to inhibit myogenic differentiation in the myogenin-expressing 10T1/2 cells. The interpretation of this observation in the context of alveolar rhabdomyosarcomas is difficult because we observe that this subtype strongly expresses both MyoD as well as myogenin, and, despite the strong expression of these factors, alveolar rhabdomyosarcomas have a high proliferative potential and are obviously prevented from terminally differentiating. It has also been shown recently that the expression of Pax-3 is sufficient to induce the expression of MyoD54,55 and myogenin55 in mice. It is tempting to speculate that the translocation-associated abnormal expression of Pax3/7-FKHR in alveolar rhabdomyosarcomas drives the strong expression of MyoD as well as myogenin in these tumors. In this regard, it would be anticipated that ectopic expression of Pax-3/FKHR would induce strong expression of MyoD and myogenin. However, we do not observe expression of MyoD in 10T1/2 cells transfected with Pax3/FKHR expression vector (P. Houghton, unpublished data).
The expression of MyoD (myf3) has been extensively examined in rhabdomyosarcomas.29-32 From these studies we observe that antibodies to MyoD can be used to diagnose rhabdomyosarcomas from other pediatric29,30 and adult31,32 neoplasms. In these studies we showed that both alveolar and embryonal rhabdomyosarcomas express MyoD. However, we did not observe marked differences in expression of MyoD between embryonal and alveolar rhabdomyosarcomas. There are no reported studies on staining for myf5 and myf6, the two remaining members of the myogenic bHLH family, in rhabdomyosarcomas. It would be interesting to study the expression of these factors in rhabdomyosarcomas, as it may shed some light on the pathological mechanisms involved in these muscle tumors. With this in mind, monoclonal antibody reagents to myf5 and myf6 are currently being developed and characterized.
In summary, we show that there is a strong association in the staining for myogenin in tumors of the alveolar histological subtype. We propose that alveolar rhabdomyosarcomas are clonally derived from myogenin-positive progenitor cells, whereas embryonal rhabdomyosarcomas may be derived from a block in myogenesis before the expression of myogenin. Therefore, the low expression of myogenin in embryonal rhabdomyosarcomas may be associated with phenotypic differentiation of the small proportion of the tumor cells. Thus antibodies to myogenin may not only be useful for diagnosing rhabdomyosarcomas from other small round and spindle cell tumors, but may help in distinguishing between embryonal and alveolar rhabdomyosarcomas by simple immunohistological techniques. Because alveolar rhabdomyosarcomas are associated with poor prognosis, our data suggest that strong expression of myogenin in rhabdomyosarcomas may be used as a marker of poor prognosis. This notion is currently being tested in a study using a large number of tumor specimens for which clinical and cytogenetic data plus patient survival data are available.
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Acknowledgements |
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Footnotes |
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Supported in part by U.S. Public Health Service grant 7R 44 CA 60198 (to P. D.); the Dean’s CUMG Development Fund, University of Arkansas Medical Sciences (to B. C.); U.S. Public Health Service grant CA 23099; and the American Lebanese and Syrian Associated Charities (to P. H.).
This work was presented at the 87th Annual Meeting of the United States/Canadian Academy of Pathology, Boston MA, March 1998 (Mod Pathol 1998, 11:14A).
Accepted for publication October 27, 1999.
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 K. T. Montone, F. G. Barr, P. J. Zhang, M. D. Feldman, and V. A. LiVolsi Embryonal and Alveolar Rhabdomyosarcoma of Parameningeal Sites in Adults: A Report of 13 Cases International Journal of Surgical Pathology, February 1, 2009; 17(1): 22 - 30. [Abstract] [PDF]
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 J.-J. Hu, A. Ambrus, T. W. Fossum, M. W. Miller, J. D. Humphrey, and E. Wilson Time Courses of Growth and Remodeling of Porcine Aortic Media During Hypertension: A Quantitative Immunohistochemical Examination J. Histochem. Cytochem., April 1, 2008; 56(4): 359 - 370. [Abstract] [Full Text] [PDF]
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 J. S. Chamberlain, J. Metzger, M. Reyes, D. Townsend, and J. A. Faulkner Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma FASEB J, July 1, 2007; 21(9): 2195 - 2204. [Abstract] [Full Text] [PDF]
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 M. Wachtel, T. Runge, I. Leuschner, S. Stegmaier, E. Koscielniak, J. Treuner, B. Odermatt, S. Behnke, F. K. Niggli, and B. W. Schafer Subtype and Prognostic Classification of Rhabdomyosarcoma by Immunohistochemistry J. Clin. Oncol., February 10, 2006; 24(5): 816 - 822. [Abstract] [Full Text] [PDF]
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 H Caldas, M P Holloway, B M Hall, S J Qualman, and R A Altura Survivin-directed RNA interference cocktail is a potent suppressor of tumour growth in vivo J. Med. Genet., February 1, 2006; 43(2): 119 - 128. [Abstract] [Full Text] [PDF]
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 P. R.J. Bois, K. Izeradjene, P. J. Houghton, J. L. Cleveland, J. A. Houghton, and G. C. Grosveld FOXO1a acts as a selective tumor suppressor in alveolar rhabdomyosarcoma J. Cell Biol., September 12, 2005; 170(6): 903 - 912. [Abstract] [Full Text] [PDF]
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 N J Sebire and M Malone Myogenin and MyoD1 expression in paediatric rhabdomyosarcomas J. Clin. Pathol., June 1, 2003; 56(6): 412 - 416. [Abstract] [Full Text] [PDF]
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E. M. Brey, Z. Lalani, C. Johnston, M. Wong, L. V. McIntire, P. J. Duke, and C. W. Patrick Jr. Automated Selection of DAB-labeled Tissue for Immunohistochemical Quantification J. Histochem. Cytochem., May 1, 2003; 51(5): 575 - 584. [Abstract] [Full Text] [PDF]
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