—  SPECIALTY CONFERENCE  —

BONE AND SOFT TISSUE PATHOLOGY

Case 4 - Alveolar Rhabdomyosarcoma

David M. Parham
Arkansas Children's Research Hospital
Little Rock, Arkansas


Click on each slide thumbnail image for an enlarged view
Clinical History:
This 14 year-old girl presented with persistent cough, fatigue, weight loss, anorexia, and headache of 2 months duration. Laboratory results included a hemoglobin of 106 g/L, leukocyte count of 8.8 X 109/L, platelet count of 335 X 109/L, serum lactate dehydrogenase of 7975 u/L, and a serum uric acid of 416 mol/L. Computed tomography of the chest and abdomen revealed a large, left-sided, pleural-based soft tissue mass associated with a large pleural effusion, compression of the left lung, and right-sided mediastinal displacement was found on computed tomography. Retroperitoneal adenopathy involving the celiac axis and associated with massive ascites was noted in the abdomen. Cytologic examination of the pleural fluid revealed malignant undifferentiated cells (Figure 1). No metaphases were obtained on cytogenetic studies. Bone marrow examination was negative for tumor. Because of the critical condition of the patient, therapy was begun based on cytologic findings, with expert consultation.

Six months after therapy, the patient had a pleural relapse. Pleural biopsy revealed a primitive small cell tumor (Figure 2), with immunohistochemical positivity for kappa light chain and CD20 and negativity for desmin.


Case 4 - Figure 1 - Cytocentrifuge preparation of pleural fluid, containing a blast with round nucleus, ropy chromatin, minimal deep blue cytoplasm, and prominent cytoplasmic vacuoles.
Case 4 - Figure 2 - Pleural biopsy of initial recurrence. The tumor comprises diffuse sheets of small cells with round, hyperchromatic nuclei, minimal cytoplasm, and no obvious features of differentiation.
Case 4 - Figure 3 - Pleural biopsy of second relapse. After bone marrow transplantation, the cancer recurred and contains fibrous septa subtending nests of tumor cells with obvious rhabdomyoblastic differentiation (intensely eosinophilic cytoplasm. Prominent clefting artefact can be seen at the periphery of the nests, adjacent to alignment of the tumor cells on the edge of the fibrous septa.

Polyphenotypia, or the formation of proteins and organelles characteristic of diverse cell types, constitutes a well-known trait of some small cell malignancies. Well-characterized examples include desmoplastic small cell tumors, which co-express epithelial, neural, and mesenchymal proteins,17  and ectomesenchymomas, which combine myogenic and neural phenotypes.2  Among more familiar embryonal neoplasms of childhood, teratoid variants of Wilms tumor and hepatoblastoma occasionally exhibit a bewildering array of cell lineages, making them difficult to distinguish from germ cell tumors.5, 12  Thus, the rare occurrence of unexpected immunostaining results in relatively common childhood cancers, like rhabdomyosarcoma, should come as no surprise to the seasoned pathologist.

Rhabdomyosarcomas can be operationally defined as lesions comprised of primitive cells with a tendency toward myogenic differentiation. Histologically, this tendency manifests itself by the variable content of rhabdomyoblasts in these lesions. We recognize these cells by their brightly eosinophilic cytoplasm, occasional cross-striations, and odd appearances (strap cells, racquet cells, spider cells, tadpole cells, broken straws, etc.). However, overtly myogenic cells may appear infrequently in primitive tumors, particularly "solid" forms of alveolar rhabdomyosarcoma.22  Conversely, chemotherapeutic agents enhance this differentiation process as a consequence of expulsion of cells from the cell cycle and into either apoptosis or G0.4 

Molecularly, a series of steps involving DNA transcription defines early myogenesis. Transcription factors, known as the "MyoD" family and including MyoD, myogenin, and myf-5, comprise a group of helix-loop-helix (HLH) proteins that actively dimerize and then insert themselves into the major groove of the DNA helix at key promotor regions. Their lock-and-key action at these upstream sequences leads to initiation of transcription at the downstream coding regions, with production of RNA that encodes muscle specific proteins such as desmin, creatine kinase, and myosin.11  Forced expression of MyoD family genes in diverse cell types, including epithelium, leads to metamorphosis into muscle cells.9 

Similarly, the expression of MyoD operationally defines rhabdomyosarcomas at a molecular level.21  This genetic model explains the frequent origin of these tumors in sites devoid of skeletal muscle, such as urinary bladder and gall bladder, and it expunges the classical notion that they are "neoplasms of skeletal muscle". Expression of MyoD family proteins differs between alveolar and embryonal subtypes, with the former showing paradoxically strong expression in undifferentiated cells and the latter exhibiting a heterogeneity of expression analogous to normal embryonic myogenesis.3, 10  Two basic observations might account for this distinction: differing methylation status, and the PAX/FKHR fusions that characterize most alveolar rhabdomyosarcomas.

Methylation of cytosine molecules in upstream promotor sequences of DNA has been associated with many biological processes, including embryogenesis, aging, lyonization, imprinting, mutagenesis, and viral insertion.6  Its occurrence generally causes inactivation of gene promotion and thus decreased transcription of downstream coding regions. Methylation proceeds through carefully orchestrated steps during embryonic development, causing temporal activation and inactivation of key organogenic proteins. In embryonal rhabdomyosarcoma, partial methylation of the MyoD promotor recapitulates the status of normally developing myoblasts. In alveolar rhabdomyosarcoma, MyoD promotor status reflects total unmethylation, corresponding to the seemingly unrestrained MyoD expression.

As a possible explanation of this latter phenomenon, PAX3 appears to be a normal modulator of MyoD family expression and promotes myogenesis as an early step in the pathway of muscle differentiation. PAX proteins contain a binding site that also interacts with DNA promotor regions and initiate expression of downstream transcription of MyoD RNA.15  The aberrant PAX/FKHR fusion of alveolar rhabdomyosarcoma may thus interact with DNA transcription in an unrestrained fashion because of its unique molecular structure, mollifying inhibitory factors and resulting in a myogenic transcription program.15  Thus, alveolar rhabdomyosarcomas do not show myogenesis because they arise in muscle, but because they contain abnormal genes that happen to initiate the process as well as contribute to tumorigenesis.

Aberrant gene expression can also serve as a hypothetical framework to explain unexpected polyphenotypia in small cell neoplasms. For example, WT1, a fusion partner in desmoplastic small cell tumor, effects epithelial cell differentiation in the kidney and mesothelium and sex cord differentiation in the developing gonads.13  In the case of rhabdomyosarcoma, one should note that HLH proteins effect not only myogenesis but also for commitment into a variety of cell lineages, including neurons and B-lymphocytes. The NeuroD family of HLH proteins is express by PNETs19  and developing neurons of the CNS.20  In lymphocytes, HLH enhances B cell development.14  Although HLH proteins can exert their effects as homodimers, heterodimers (such as MyoD-NK) also form and might exert unexpected pleiotropic influences.16  The hypothetical result could be divergent differentiation.

This particular case was one of a series of three similar tumors described by Pinto et al. in 1997.18  All three tumors were originally diagnosed as B-cell neoplasms because of features such as monoclonal immunoglobulin production and CD19 positivity. In each instance, the rhabdomyosarcomatous nature of the lesion became apparent only after combination chemotherapy. In two cases, a t(2;13) was present on cytogenetic evaluation, confirming the diagnosis of solid alveolar rhabdomyosarcoma. Of particular note is the occasional description of B-cell neoplasms with an identical translocation,1  raising questions about their true identity. The possibility of a legal action in difficult cases because of a mistaken diagnosis based on unusual ancillary features should be of great concern. This topic was discussed in a editorial by Dehner and a subsequent series of letters.7, 8 

Histologic findings
The initial diagnostic material consisted of pleural fluid cytocentrifuge preparations, containing malignant, singly dispersed cells with the features of blasts (Figure 1). These cells exhibited high nucleocytoplasmic ratios, ropy chromatin, and purple cytoplasm on Wright-Giemsa stained slides. Cytoplasmic vacuoles were prominent. Based on the cytologic features of the lesion and because of the emergent nature of the case, a diagnosis of B-cell lymphoma was rendered. The tumor responded to therapy.

At the time of the initial recurrence, a pleural biopsy was performed. The histologic features of the lesion were those of an undifferentiated small cell neoplasm, containing diffuse sheets of primitive cells (Figure 2). The tumor cells contained round, hyperchromatic nuclei and minimal cytoplasm. Immunohistochemistry and flow cytometry confirmed the initial impression of a B-cell neoplasm.

After bone marrow transplantation, the tumor again responded to therapy. Unfortunately, another pleural relapse occurred, and the lesion was rebiopsied. At this point, the tumor contained prominent fibrous septa that circumscribed nests of tumor cells with brightly eosinphilic cytoplasm, indicative of rhabdomyoblastic differentiation. The cells formed cohesive central clusters surrounded by clefts, which in turn abutted rows of cells aligning the septa (Figure 3). This post-therapy differentiation confirmed the diagnosis of alveolar rhabdomyosarcoma.

Differential diagnosis (of initial biopsy)

  • Ewing sarcoma/primitive neuroectodermal tumor/Askin tumor
  • Pleuropulmonary blastoma
  • Malignant lymphoma, small non-cleaved (Burkitt type)
  • Acute myeloblastic leukemia
  • Alveolar rhabdomyosarcoma
  • Synovial sarcoma, poorly differentiated
  • Malignant peripheral nerve sheath tumor, juvenile
  • Desmoplastic small round cell tumor

Final diagnosis

  • Alveolar rhabdomyosarcoma

Acknowledgement
I thank Dr. Alfredo Pinto, Alberta Children's Hospital, for his generosity in sharing this case material with the USCAP and me.

References

  1. Adami F, Sancetta R, Trentin L, et al. The pediatric rhabdomyosarcoma translocation (2;13)(q35; q14) in B-prolymphocytic leukemia. Leukemia 1993;7:1676-1678.
  2. Boue DR, Parham DM, Webber B, Crist WM, Qualman SJ. Clinicopathologic study of ectomesenchymomas from Intergroup Rhabdomyosarcoma Study Groups III and IV. Pediatr Devel Pathol 2000;3:290-300.
  3. Chen B, Dias P, Jenkins JJ, Savell VH, Parham DM. Methylation alterations of the MyoD1 upstream region are predictive of subclassification of human rhabdomyosarcomas. Am J Pathol 1998;152:1071-1079.
  4. Coffin CM, Rulon J, Smith L, Bruggers C, White FV. Pathologic features of rhabdomyosarcoma before and after treatment: a clinicopathologic and immunohistochemical analysis. Mod Pathol 1997;10:1175-1187.
  5. Conrad RJ, Gribbin D, Walker NI, Ong TH. Combined cystic teratoma and hepatoblastoma of the liver: Probable divergent differentiation of an uncommitted hepatic precursor cell. Cancer 1993;72:2910-2913.
  6. Cooney CA. Are somatic cells inherently deficient in methylation metabolism? A proposed mechanism for DNA methylation loss, senescence and aging. Growth Dev Aging 1993;57:261-273.
  7. Dehner LP. On trial: a malignant small cell tumor in a child: four wrongs do not make a right {see comments}. Am J Clin Pathol 1998;109:662-668.
  8. Dehner LP. The author's reply. Am J Clin Pathol 1999;111:426-427.
  9. Dekel I, Magal Y, Pearson-White S, Emerson CP, Shani M. Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene. New Biol 1992;4:217-224.
  10. Dias P, Chen B, Dilday B, et al. Strong immunostaining for myogenin in rhabdomyosarcoma is significantly associated with tumors of the alveolar subclass. Am J Pathol 2000;156:399-408.
  11. Dias P, Dilling M, Houghton P. The molecular basis of skeletal muscle differentiation. Sem Diag Pathol 1994;11:3-14.
  12. Fernandes ET, Parham DM, Ribeiro RC, Douglass EC, Kumar AP, Wilimas J. Teratoid Wilms' tumor: the St Jude experience. J Pediatr Surg 1988;23:1131-1134.
  13. Hastie ND. Wilms' tumour gene and function. Curr Opin Genet Dev 1993;3:408-413.
  14. Kadesch T. Helix-loop-helix proteins in the regulation of immunoglobulin gene transcription. {Review} {46 refs}. Immunology Today 1992;13:31-36.
  15. Khan J, Bittner ML, Saal LH, et al. cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene. Proc Natl Acad Sci USA 1999;96:13264-13269.
  16. Murre C, McCaw PS, Vaessin H, et al. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 1989;58:537-544.
  17. Ordonez NG. Desmoplastic small round cell tumor: II: an ultrastructural and immunohistochemical study with emphasis on new immunohistochemical markers. {Review} {98 refs}. Am J Surg Pathol 1998;22:1314-1327.
  18. Pinto A, Tallini G, Novak RW, Bowen T, Parham DM. Undifferentiated rhabdomyosarcoma with lymphoid phenotype expression. Med Pediatr Oncol 1997;28:165-170.
  19. Rostomily RC, Bermingham-McDonogh O, Berger MS, Tapscott SJ, Reh TA, Olson JM. Expression of neurogenic basic helix-loop-helix genes in primitive neuroectodermal tumors. Cancer Res 1997;57:3526-3531.
  20. Schwab MH, Bartholomae A, Heimrich B, et al. Neuronal basic helix-loop-helix proteins (NEX and BETA2/Neuro D) regulate terminal granule cell differentiation in the hippocampus. Journal of Neuroscience 2000;20:3714-3724.
  21. Scrable H, Witte D, Shimada H, et al. Molecular differential pathology in rhabdomyosarcoma. Genes Chromosom Cancer 1989;1:23-35.
  22. Tsokos M, Webber B, Parham D, et al. Rhabdomyosarcoma: a new classification scheme related to prognosis. Arch Pathol Lab Med 1992;116:847-855.