Strong immunostaining for myogenin in rhabdomyosarcoma is significantly associated with tumors of the alveolar subclass

Abstract

Rhabdomyosarcomas are a heterogeneous group of tumors with respect to their molecular basis, degree of differentiation, histology, and clinical behavior. Because of the wide variation of tumor morphology, it is often difficult to distinguish between the distinct subtypes of rhabdomyosarcomas. By using cryosections of tumor specimens and immunohistochemistry, in the present study we show that strong expression of myogenin in rhabdomyosarcoma is associated with alveolar histology (P = <0.0001, Fisher's exact test). Although staining for myogenin was observed in 22 of 26 rhabdomyosarcomas, all alveolar rhabdomyosarcomas (nine of nine) showed high levels of staining for myogenin, as defined by the frequency and intensity of staining of the tumor cells. The staining pattern suggests that the tumor cells are clonally derived from myogenin-positive progenitor cells. In contrast, most embryonal rhabdomyosarcomas (13 of 15) were either negative or showed a low level of staining for myogenin. In these tumors a larger proportion of tumor cells were distinctly negative for myogenin. Six of seven alveolar rhabdomyosarcomas that strongly stained for myogenin were also positive for Pax3-7/Forkhead (FKHR) by polymerase chain reaction/reverse transcriptase-polymerase chain reaction. One of two embryonal rhabdomyosarcomas that strongly stained for myogenin was retrospectively found to be positive for Pax3/FKHR transcripts. Quantitative analysis for myogenin by Western blotting using a smaller subset of rhabdomyosarcomas revealed that in general there was a good correlation between immunohistochemical staining and Western blotting (P = 0.01, Pearson Correlation), although the former technique was more sensitive for detecting tumors with low levels of the protein. On average, alveolar rhabdomyosarcomas expressed at least threefold more myogenin than embryonal rhabdomyosarcomas. Our data show that staining for myogenin will be a simple, rapid, and accurate adjunct for distinguishing between alveolar and embryonal rhabdomyosarcomas. We propose that embryonal rhabdomyosarcomas result from an early block in myogenesis, before the expression of myogenin. In contrast, we propose that alveolar rhabdomyosarcomas either originate from a late block in myogenesis (after expression of myogenin) or that the pathological mechanisms involved in these neoplasms also induce strong expression of this protein.

Figure 1.

Western blot analysis of myogenin expression in various small round cell tumor lines using the anti-myogenin monoclonal antibody. The antibody only reacts with a protein band corresponding to the molecular mass of myogenin (approximately 34 kd) in the Rh30 rhabdomyosarcoma cell lysate. All other small round cell tumor lysates were negative. Rh30, alveolar rhabdomyosarcoma cell line; PFSK-1A, primitive neuroectodermal tumor cell line; EB2, lymphoma cell line; SKNSH, neuroblastoma cell line; SJSA-1, Ewing’s sarcoma cell line.

Figure 2.

Representative staining for myogenin in embryonal and alveolar rhabdomyosarcoma. The embryonal rhabdomyosarcoma (top panels) in this figure was less than 10% positive for myogenin and was scored as (+), whereas most tumor cells (85–95%) in the alveolar rhabdomyosarcoma tumor (bottom panels) were homogeneously strongly positive for myogenin. This tumor was scored as (+++). Left: Lower magnification (original magnification, ×200). Right: Higher magnification (original magnification, ×400). ABC staining, methyl green counterstain.

Figure 3.

Western blot analysis for myogenin expression in a smaller subset of rhabdomyosarcoma specimens. Four of six alveolar rhabdomyosarcomas (lanes 8, 10, 11, and 12) were strongly positive for myogenin. One alveolar rhabdomyosarcoma (lane 7) was negative, and one alveolar tumor (lane 9) was weakly positive for myogenin. Three of six embryonal rhabdomyosarcomas (lanes 3, 4, and 6) were negative for myogenin, whereas two of six (lanes 2 and 5) were weakly positive. One embryonal rhabdomyosarcoma (lane 1), a posttreatment tumor, was strongly positive for myogenin. The blot was incubated with anti-myogenin monoclonal antibody. Reactivity was detected using a horseradish peroxidase-conjugated secondary antibody and visualized using a chemiluminescent substrate and exposure to photographic films.