Fine-Needle Aspiration Biopsy Diagnosis of Rhabdomyosarcoma: Cytologic, Histologic, and Ultrastructural Correlations Mohammed Akhtar, M.D., F.c.A.P., Muhammad Ashraf Ah, M.D., F.R.C.P.G., F.c.A.P., Mohammed Bakry, B.s., M.B., Mahmuda Hug, A.I.M.L.s., and Kwasi Sackey, M.D.
A series of I5 cases of rhabdomyosarcoma diagnosed by fineneedle aspiration biopsy (FNAB) and confirmed by histopathology is reviewed. Cytologically, the tumors were composed of a variable mixture of cells, which according to the degree of differentiation were categorized as early, intermediate, or late rhabdomyoblasts. Histologically, the tumors were divided into embryonal 9, monomorphic round cell 4, and alveolar rhabdomyosarcoma 2. Comparison of histological and cytological features revealed that embryonal types were composed mainly of early rhabdomyoblasts. Recognition of these patterns may be helpful in FNAB diagnosis of rhabdomyosarcoma. Diagn Cytopathol 1992;8:465474. 0 1992 Wiley-Liss, lnc.
Key Words: Rhabdomyoblast; FNAB; Ultrastructure; Embryonal; Alveolar; Round cell
Fine-needle aspiration biopsy diagnosis of rhabdomyosarcoma has been described in several previously published case However, to the best of our knowledge, a detailed study describing the spectrum of morphologic features of RMS in FNAB smears has not yet been described. The purpose of this study is to review our experience with a series of 15 rhabdomyosarcomas in which FNAB diagnosis was further confirmed by histological examination. The FNAB findings in these cases are correlated with histologic and ultrastructural features in order to understand and elucidate the spectrum of cytomorphologic patterns in rhabdomyosarcoma.
Materials and Methods Rhabdomyosarcoma (RMS) is one of the most common malignant tumors in children. In many cases, histological diagnosis of this tumor is often difficult because of poor differentiation of tumor cells. They may be confused with other types of small round cell tumors such neuroblastoma, Ewing’s sarcoma, neuroectodermal tumor, Wilms’ tumor, and malignant lymphoma. Fine-needle aspiration biopsy may be used to diagnose a variety of benign and malignant neoplasms. In children, most of the malignant neoplasms belong to the category of round cell malignancies. Fine-needle aspiration biopsy diagnosis of these tumors may be difficult because of significant morphologic overlap. However, several recent studies have described morphological features that may help in making these distinctions on the basis of aspiration cytology. 2-8
’
Received September 4, 1991. Accepted January 15, 1992. From the Department of Pathology and Laboratory Medicine and Department o f Oncology, King Faisal Specialist Hospital, and Research Centre, Riyadh, Saudi Arabia. Address reprint requests to Dr. Mohammed Akhtar, Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia. (c)
1992 W I L t > - L I S S . INC
All cases of rhabdomyosarcoma diagnosed by FNAB between January 1980 and December 1990 were identified and of these, a group of cases in which the FNAB diagnosis was further confirmed by histologic examination, were selected for review. Of the 31 cases identified, nine were excluded because the cellular material in the FNAB smears was inadequate for detailed evaluation. Another seven cases were excluded because the material for immunohistochemistry was not available. The remaining 15 cases form the basis of this study (Table I). Fine-needle aspiration biopsies were performed by using 22-25-gauge needles with or without aspiration. Smears were air-dried and stained by Diff-Quik stain. For immunohistochemical studies, a portion of the material was placed in vials containing tissue culture medium (RPMI-I640 Hazelton Cat #50178); 0.1-0.2 ml of the suspension was used to obtain cytospin preparations using 500 RPM for 5 min. These slides were stored in a refrigerator at 4°C for a maximum of 5 days. After fixation in ice-cold acetone for 10 mins, immunoperoxidase staining was performed using ABC technique. The electron microscopy part of the FNAB sample was fixed in 3% glutaDiugnostic Cytoputhology, Vol 8. No 5
465
AKHTAR ET AL. Table I. Clinicopathologic Features of 15 Cases of RMS Case no.
1 2 3 4
5
6 7 8 9 10 I1 12 13 14
15
Location of FNAB
Electron microscopy
I m m unoperoxiduse staining on FNAB
I m m unoperoxidase stuining of titsue
Chest wall Elbow Left groin Left neck Left axilla Rt. upper arm Neck and R t . maxilla Left orbit Gluteal Mediastinurn Abdomen ParaInguinal areas Upper arm and lung
Yes No No Yes Yes Yes No No No Yes No Yes Yes Yes No
Yes
Yes Yes Y es Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Age and sex 9M 13F 19F 9M 1OM 1M 9M 2M 11M
5M 4M 2F 2/12M 15F SM
raldehyde for at least 1 hr and then processed according to the technique described previously. l 4 Tissue biopsy specimens were fixed in 10% buffered formaldehyde solution and embedded in paraffin in a routine fashion. Sections were stained with hematoxylin and eosin stain. For immunohistochemical studies, sections were stained using antibodies against vimentin (DAKO) Desmin (BIOGENICS) and myoglobin (DAKO) using ABC technique.
Results Detailed review of aspiration smears in our cases revealed that the neoplasms were composed of a variable mixture of tumor cells that showed marked variation in size and shape. However, based on the degree of apparent myogenic differentiation, these cells were divided into three categories, namely, early, intermediate, and late rhabdomyoblasts. Early rhabdomyoblasts were round, undifferentiated cells with large, irregular, single nuclei and scanty basophilic cytoplasm (Figs. C-1, C-2). Intermediate rhabdomyoblasts had relatively abundant pale staining variably vacuolated cytoplasm and one or more irregular nucleus with occasional nucleolus (Fig. C-3). Late rhabdomyoblasts contained abundant cytoplasm that stained grayish blue and opaque. These cells varied from round to markedly elongated resembling myotubules (Figs. C-4, C-5).Some of these cells had localized inclusion-like grayish blue areas within the cytoplasm (Fig. C-6). Some of the intermediate and late rhabdomyoblasts were extremely large with markedly pleomorphic nuclei. The relative proportion of various cell types varied considerably from one case to another. Whereas a majority of the tumors contained all three types of cells, in some only two cell types, namely, early and intermediate rhabdomyoblasts, were recognized (Table 11). The tumor cells were loosely arranged without any specific pattern. Occasionally, early rhabdomyoblasts were arranged in small 466
Diagno.rtic Cyioputhology, Voi 8,No 5
No NO
No No No Yes No Yes NO NO
Yes
No Yes NO
tight cell clusters; however, no cell clusters composed of intermediate or late rhabdomyoblasts were encountered (Fig. C-2). Histologic examination of the tumors together with electron microscopy and immunohistochemistry enabled the diagnosis of rhabdomyosarcoma to be made either by identification of rhabdomyoblasts or by characteristic growth pattern such as seen in alveolar rhabdomyosarcoma. These tumors were further subdivided into the following morphologic subtypes: embryonal RMS, 9; monomorphic round cell RMS 4; and alveolar RMS, 2. l 5 Comparison of histopathologic appearance of tumors with cytomorphologic features in aspiration smears indicated a close correlation. Thus embryonal rhabdomyosarcomas, which on histological examination were found to contain large numbers of cells with obvious myogenic differentiation, tended to reveal relatively frequent intermediate and late rhabdomyoblasts (Figs. C-4-C-6). By contrast, alveolar RMS and monomorphic round cell RMS were composed of relatively undifferentiated small round cells (Figs. C-1, C-2). Evidence of myogenic differentiation in these tumors was found only after careful examination of several sections. Corresponding FNAB smears revealed large numbers of early rhabdomyoblasts with variable scattering of intermediate cells and only rare late rhabdomyoblasts (Table 11). Ultrastructurally, early rhabdomyoblasts contained small amount of cytoplasm with large numbers of ribosomes, few mitochondria, and occasional profiles of rough endoplasmic reticulum (Fig. 1). Intermediate rhabdomyoblasts contained relatively abundant cytoplasm rich in organelles such as mitochondria, profiles of rough endoplasmic reticulum and variable numbers of lyosomes and aggregates of glycogen (Figs. 2, 3). Some of these cells in addition revealed small parallel aggregates of thick (myosin) and thin (actin) filaments (Fig. 4). Late rhab-
RHABDOMYOSARCOMA DIAGNOSIS Table 11. Cytologic, Histologic, and Ultrastructural Correlations
Case no.
2 4 7 9 10 11
12 13
I5 I 14 3 5 6 8
Histologic subtype
Immunohistochemical findingv'
Embryonal Embryonal Embryonal Embryonal Embryonal Embryonal Embryonal Embryonal Embryonal Alveolar Alveolar Monomorphic Monomorphic Monomorphic Monomorphic
"V = vimentin, D = desmin, M
V+D+M+ V+D+M+ V+D+MV+D+M+ V+D+M+ V+D+M+ V+D+M+ V+D+MV+D+M+ V+D+MV+D+MV+D+MV+D+MV+D+M+ V+D+M=
in
15 Cases of RMS
Myogen ic diffcrentrotion
Cytologic types of rhabdornyoblasts Early ++i
++ ++ + + + ++ ++ ++++ ++++ +++ ++++ ++++ +++
In term edia te
+ ++ + +
++ ++ ++ ++ ++ + + ++ + + +
Lute t
+ ++ +++ ++ ++ t + f i
ot7
Not Ye\ Not Not Ye\ Not Ye\ Ye\ Not Yes Not Not Not Ye\ Not
EM done
done done done
done present done pre\ent
done
myoglobin.
Fig. 1. Electronmicrograph depicting early rhabdornyoblasts in FNAB from RMS. The tumor cells are undifferentiated with not clear evidence of myogenic differentiation (Uranyl acetate and lead citrate stain, x 12,000). Diagnostic Cytopathology, Vol 8, No 5
461
Fig. C-1A
Fig. C-1B
Fig. C-ZA
Fig. C-ZB
Fig. C-3A
Fig. C-3B
Figs. C - 1 4 - 3 . Fig. C-1. A. FNAB smear, featuring early rhabdomyoblasts with large nuclei and relatively scanty cytoplasm (Diff-Quik stain, X 150). B. Histologic section from a monomorphic round cell RMS composed of undifferentiated round cells (Diff-Quik stain, X 150). Fig. C-2. A. Early rhabdomyoblasts forming small clusters in an alveolar RMS (Diff-Quik stain, X 150). B. Histologic section showing a representative area i n alveolar RMS. The tumor cell\ are small and undifferentiated and tend to form alveolar spaces (Hematoxylin & Eosin stain, x 100). Fig. C-3. A. Aspiration smear showing loosely arranged intermediate rhabdomyoblasts (Diff-Quik stain, X 150). B. Higher magnification photomicrograph showing intermediate rhabdomyoblasts with abundant pale staining cytoplasm. Some of the cells have multiple nuclei (Diff-Quik stain, X 250).
Fig. C-4A
Fig. C-4B
Fig. C-5A
Fig. C-5B
Fig. C-6A
Fig. C-6B
Figs. C - 4 4 - 6 Fig. C-4. A. Aspiration cells depicting four late rhabdomyoblasts. The cytoplasm is abundant, grayish blue and uniformly opaque (Diff-Quik stain, X 200). B. Histologic sections from embryonal RMS showing cells with abundant, diffusely eosinophilic cytoplasm indicating myogenic differentiation (Hematoxylin and Eosin stain, x 150). Fig. C-5. A. Aspiration smear showing several elongated late rhabdomyoblasts from embryonal RMS (Diff-Quik stain, X 200). B. Corresponding histologic section showing well-differentiated rhabdornyoblasts (Hematoxylin and Eosin stain, X 150). Fig. C-6. A. A late rhabdomyoblast in aspiration smear containing an elongated inclusion-like area within the cytoplasm (Diff-Quik stain, X 250). B. Histologic section showing several rhabdomyoblasts with eosinophilic inclusion-like areas of myogenic differentiation (Hematoxylin and Eosin stain, x200).
Fig. 2. Electronmicrograph featuring intermediate rhabdomyoblasts. The cytoplasm is relatively abundant with various organelles (Uranyl acetate and lead citrate stain, x 14,000).
Fig. 3. Another intermediate rhabdomyoblast with abundant cytoplasm, which X 5,700).
470
Diugnomt Cyropofhology.Vol 8. No 5
IS extremely
rich in organelles (Uranyl acetate and lead citrate stain,
RHABDOMYOSARCOMA DIAGNOSIS
domyoblasts contained cytoplasm similar to that seen in intermediate rhabdomyoblasts. However, these cells in addition contain larger aggregates of thick and thin filaments with varying degrees of sarcomeric organization. These zones of myogenic differentiation may involve small localized areas of the cytoplasm or may virtually replace the entire cytoplasm with virtual exclusion of other organelles (Fig. 5).
Discussion FNAB diagnosis of RMS is primarily dependent upon demonstration of cells that could be clearly identified as rhabdomyoblasts. Late rhabdomyoblasts with their distinctive opaque cytoplasm may be identified with relative certainty. Intermediate rhabdomyoblasts with relatively abundant pale cytoplasm may also be tentatively recognized, but their precise identity may only be established by immunocytochemistry and/or ultrastructural examination. Early rhabdomyoblasts are morphologically undifferentiated cells that closely mimic similar cells in other types of round cell malignancies. These cells are generally without any demonstrable ultrastructural features of myogenic differentiation. Immunocytochemistry, in contrast, is much more likely to provide a positive identification since most of these cells stain positive for vimentin and desmin. The role of electron microscopy in the diagnosis of RMS IS well-established. I6-l8 The material for electron microscopy may be derived from a tissue biopsy or fineneedle aspiration biopsy. The minimum requirement for ultrastructural diagnosis is the demonstration of actin and myosin (thick) filaments or recognition of ribosome-myosin complex. More differentiated cells may show parallel organization of sarcomeres with formation of M-lines and Z bands. Ultrastructural evidence of myogenic differentiation may be easily obtained in FNAB material from embryonal rhabdomyosarcoma; however, it may be difficult in other types of RMS.2Q^2‘Tumors with strong immunohistochemical evidence of rhabdomyomatous differentiation (usually strong desmin positivity) but without ultrastructural features of muscle differentiation represent a difficult diagnostic dilemma. Some authors have characterized such tumors as RMS-like tumors. 2 1 Others argue that failure to demonstrate organized myofilaments does not exclude the possibility of poorly differentiated rhabdomyosarcoma. 2o In two cases ultrastructural evidence of myogenic differentiation could not be found, although tumor cells in both were strongly positive for desmin. One of these had morphological features characteristic of alveolar RMS. Admittedly, this discrepancy may be explained on the basis of sampling error. However, we believe that a more plausible explanation maybe that desmin is expressed within rhabdomyoblasts before any myofilaments can be recognized.
A large number of immunocytochemical markers for diagnosis of RMS have been studied. These include desmin, skeletal muscle specific actin, muscle specific actin, myoglobin, myosin, troponin, creatine kinase, Z-band protein, and titen. 14*21-24However, the most widely used marker is desmin, which is an intermediate filament protein located in close association with Z-band material. The sensitivity and specificity of desmin in the diagnosis of RMS have been the subject of numerous studies. It is now generally agreed that Desmin is an extremely sensitive and a fairly reliable marker for smooth and striated muscle differentiation. 23-25 Smooth muscle neoplasms are rare in children and therefore do not cause any significant diagnostic difficulty with rhabdomyosarcoma. Other conditions in which desmin positivity may occasionally be seen include Wilms’ tumor, malignant fibrous histiocytoma, glial tumors, malignant Schwannoma, malignant melanoma, malignant mesothelioma, fibromatosis, renal rhabdoid tumor, myositis ossificans, and embryonal sarcoma of liver. 22-23 Most of these lesions, however, rarely pose diagnostic difficulty with rhabdomyosarcorna in children. Nevertheless, we believe that full spectrum of desmin reactivity in various benign and malignant neoplasms is yet to be determined. Consequently, it is advisable to confirm cytologic and histologic diagnosis of rhabdomyosarcoma by immunohistochemistry as well as electronmicroscopy in as many cases as possible. Correlation of cytologic, histologic, and ultrastructural findings in our study indicates that early, intermediate, and late rhabdomyoblasts reflect various stages in the differentiation of tumor cells. Early rhabdomyoblasts are primitive cells in which there is no morphologic evidence of myogenic differentiation. However, on immunocytochemistry, most of these cells stain with desmin indicating a commitment to such a differentiation. Ultrastructurally, these cells are devoid of actin and myosin filaments. The intermediate myoblasts are comparatively more differentiated but still do not reveal any definite evidence of myogenic differentiation at light microscopic level. Ultrastructurally, however, a few small cytoplasmic aggregates of thin and thick filaments may be seen, although they are not arranged to form distinct bands. Late rhabdomyoblasts are cells that show obvious myogenic differentiation at light microscopy in the form of dense, opaque areas within the cytoplasm. These areas involve variable portion of the cytoplasm. Ultrastructural appearance of these areas corresponds to the presence of large bundles of thick and thin filaments with sarcomeric arrangements. When myogenic differentiation is limited to a small localized area within the cytoplasm, it may appear as an inclusionlike area. These inclusions are also composed of thick and thin filaments with Z-band formation. In a recent study rhabdoid-like inclusions have been described in some of the rhabdomyosarcomas. 27 These are ultrastructurally Diagnostic Cyropathology, Vol 8, NO 5
47 1
Fig. 4. Higher power electron micrograph revealing small bundles of thick and thin filaments in an intermediate rhabdomyoblart (Uranyl acetate and lead citrate stain, x 25,000).
Fig. 5. A-C. Late rhabdomyoblasts depicting various patterns of myogeriic differentiation (Uranyl acetate and lead citrate stain, A : X 14,000; B: x 12,000;c: x Is,oao).
472
Diugriostie Cytoputhology. Vol 8. No 5
RHABDOMYOSARCOMA DIAGNOSIS
Fig. 5R-C.
Diagmrtic Cytopathology, Yo1 8, No 5
413
AKHTAR ET AL.
composed of aggregates of intermediate filaments and are thus different from the myogenic inclusions seen in our study. Rhabdomyosarcoma is not the only neoplasm in which rhabdomyoblasts may be present. Focal rhabdomyoblastic differentiation occurs in a variety of malignant neoplasms such as malignant mixed tumor of the uterus and ovary, carcinosarcoma of breast, teratoma, pulmonary blastoma, hepatoblastoma, and nephroblastoma. In addition, rhabdomyoblastic differentiation is also a feature of various neuroectodermal neoplasms notably malignant Schwannoma (malignant Triton tumor), ectomesenchymoma and medullo-epithelioma. ** Fine-needle aspiration biopsy smears from these neoplasms may be extremely difficult to interpret. Evaluation of clinical features may help to exclude some of these tumors. Precise FNAB diagnosis, however, will depend upon recognition of the cellular patterns of the parent tumor. Fortunately, with the exception of Wilms’ tumor, these neoplasms are rare. We have encountered rhabdomyoblasts in the aspiration smears from some of the Wilms’ tumors that we have studied. In these cases, recognition of the typical cellular components namely, blastemal, tubular, and stromal patterns served to establish FNAB diagnosis of Wilms’ tumor with rhabdomyomatous elements. In summary, FNAB findings in a series of 15 cases of RMS are described and correlated with histologic and ultrastructural features. It is suggested that embryonal rhabdomyosarcoma with many late and intermediate rhabdomyoblasts may easily be diagnosed on FNAB. Undifferentiated RMS, in contrast, would generally require immunohistochemistry and/or electron microscopy.
’
References 1. Triche HM, Askin FB, Kissane JM. Neuroblastoma Ewing’s sarcoma and the differential diagnosis of small round blue cell tumors.
2.
3.
4. 5.
6.
7.
In: Finegold M ed. Pathology of neoplasia in children and adolescents. Major Problems in Pathology. Philadelphia: WB Saunders, 1986;18:14S-95. Akhtar M, Ali MA, Sabbah R, Bakry M, Nash JE. Fine needle aspiration biopsy diagnosis of round cell malignant tumors of childhood: a combined light and electron microscopic approach. Cancer 1 985:5 5: 1805- 17. Akhtar M, Ali MA, Sabbah R. Aspiration cytology of Ewing’s sarcoma: light and electron microscopic correlations. Cancer 1985; 56:2051-60. Miller JR, Bottles K , Abele JS, Becksted JH. Neuroblastoma diagnosed by fine needle aspiration biopsy. Acta Cytol 1985;29:461-8. Akhtar M, Ali MA, Sabbah R, Bakry M, Sackey K , Nash EJ. Aspiration cytology of neuroblastoma: light and electron microscopic correlations. Cancer 1986;57:797-803. Silverman JF, Dabbs DJ. Ganick DJ, Holbrook CT, Geisinger KR. Fine needle aspiration cytology of neuroblastoma including peripheral neuroectodermal tumor with immunohistochemical and ultrastructural confirmation. Acta Cytol 1988;32:367-76. Valkov L, Boijikin B. Fine needle aspiration biopsy of abdominal and retroperitoneal tumors in infants and children. Diagn Cytopatho1 1987:3:129-33.
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8 Akhtar M, Ali MA, Sackey K, Sabbah R, Burgess A. Aspiration cytology of Wilms’ tumor: Correlation of cytologic and histologic features. Diagn Cytopathol 1989;5:269-74. 9 Mariarty AT, Nelson WA, McGahey B. Fine needle aspiration of rhabdomyosarcoma of heart: light and electron microscopic findings and histologic correlation. Acta Cytol 1990;34. 10 Pettinato G, Swanson PE, Insabato L, Chiara AD, Wick MR. Undifferentiated small round-cell tumors of childhood: the immunohistochemical demonstration in fine needle aspirates. Diagn Cytopathol 1989;s:194-9. 11 Seidal T, Walaas L, Kindblom LG, Angervall L. Cytology of embryonal rhabdomyosarcoma. A cytologic, light microscopic, electron microscopic and immunohistobhemical study of seven cases. Diagn Cytopathol 1988;4:292-9. 12 Torres V, Ferrer R. Cytology of fine-needle aspiration biopsy of primary breast rhabdomyosarcoma in an adolescent girl. Acta Cytol 1985;29:43M. 13 de-Jong AS, Van-Kessel-Van Vark M, Van-Heerde P. Fine needle aspiration biopsy diagnosis of rhabdomyosarcoma: an immunohistochemical study. Acta Cytol 1987;31:573-7. 14 Akhtar M, Bakry M, Nash EJ. An improved technique for processing aspiration biopsy for electron microscopy. Am J Clin Pathol 1986;85:57-60. 15 Dodd S, Malone M, McCulloch W. Rhabdomyosarcoma in children: a histologic and immunohistochemical study of 59 cases. J Pathol 1989;158: 13-18. 16 Erlandson RA. The ultrastructural distinction between rhabdomyosarcoma and other undifferentiated sarcomas. Ultract Pathol 1987;11 183-101. 17 Siedal T, Kindblom LG. The ultrastructure of alveolar and embryonal rhabdomyosarcoma: a correlative light and electron microscopic study of 17 cases. Acta Pathol Microbiol Immunol Scand Sect 1984; 92:23148. 18 Mierau GW, Favara BE. Rhabdomyosarcoma in children: ultrastructural study of 31 cases. Cancer 1980;46:203540. 19 Ghadially FN. Diagnostic electron microscopy of tumors, 2nd ed. London: Butterworth, 1985; 186-205. 20 Seidal T, Kindblom LG, Angervall L. Alveolar and poorly differentiated rhabdomyosarcoma: a clinicopathologic light-microzcopic ultrastructural and immunohistochemical analysis. APMIS 1988;96: 825-38. 21 Miettinen M, Rapola J. Immunohistochemical spectrum of rhabdomyosarcoma and rhabdomyosarcoma-like tumors: expression of cytokeratin and 68-KD neurofilament protein. Am J Surg Pathol 1989; 13:120-32. 22 Molenaar WM, Oosterhius JW, Oosterhius AM, Ramaeker FCS. Mesenchymal and muscle specific intermediate filaments (vimentin and desmin) in relation to differentiation in childhood rhabdomyosarcoma. Human Pathol 1985;16:83843. 23 Truong LD, Rangdaeng S, Cagle P, Ro TY, Hawkins H, Font RL. The diagnostic utility of desmin: study of 584 cases and review of literatures. Am J Clin Pathol 1990;93:305-14. 24 Leader M, Patel J, Collins M, Henry K. Myoglobin: an evaluation of its role as marker of rhabdomyosarcoma. Br J Cancer 1989;59: 106-9. 25 Ordonez NG. Antidesmin antibodies (Editorial): their use in diagnostic pathology. Am J Clin Pathol 1990;93:43&1. 26 Parhan DM, Webber B, Holt H, Williams WK, Maurer M. Inimunohistochemical study of childhood rhabdomyosarcoma and related neoplasms: results of an intergroup rhabdomyosarcoma study project. Cancer 1991:67;3072-80. 27 Kodet R, Newton WA, Hamoudi AB, Asmar L. Rhabdomyosarcomas with intermediate filament inclusions and features of rhabdoid tumors: light microscopic and immunohistochemical study. Am J Surg Pathol 1991;15:257-67. 28 Enzinger FM, Wiess SW. Rhabdomyosarcoma. In: Enzinger FM and Wiess SW, eds. Soft tissue tumors. St. Louis: C.V. Mosby, 1988:448-88.
A series of I5 cases of rhabdomyosarcoma diagnosed by fineneedle aspiration biopsy (FNAB) and confirmed by histopathology is reviewed. Cytologically, the tumors were composed of a variable mixture of cells, which according to the degree of differentiation were categorized as early, intermediate, or late rhabdomyoblasts. Histologically, the tumors were divided into embryonal 9, monomorphic round cell 4, and alveolar rhabdomyosarcoma 2. Comparison of histological and cytological features revealed that embryonal types were composed mainly of early rhabdomyoblasts. Recognition of these patterns may be helpful in FNAB diagnosis of rhabdomyosarcoma. Diagn Cytopathol 1992;8:465474. 0 1992 Wiley-Liss, lnc.
Key Words: Rhabdomyoblast; FNAB; Ultrastructure; Embryonal; Alveolar; Round cell
Fine-needle aspiration biopsy diagnosis of rhabdomyosarcoma has been described in several previously published case However, to the best of our knowledge, a detailed study describing the spectrum of morphologic features of RMS in FNAB smears has not yet been described. The purpose of this study is to review our experience with a series of 15 rhabdomyosarcomas in which FNAB diagnosis was further confirmed by histological examination. The FNAB findings in these cases are correlated with histologic and ultrastructural features in order to understand and elucidate the spectrum of cytomorphologic patterns in rhabdomyosarcoma.
Materials and Methods Rhabdomyosarcoma (RMS) is one of the most common malignant tumors in children. In many cases, histological diagnosis of this tumor is often difficult because of poor differentiation of tumor cells. They may be confused with other types of small round cell tumors such neuroblastoma, Ewing’s sarcoma, neuroectodermal tumor, Wilms’ tumor, and malignant lymphoma. Fine-needle aspiration biopsy may be used to diagnose a variety of benign and malignant neoplasms. In children, most of the malignant neoplasms belong to the category of round cell malignancies. Fine-needle aspiration biopsy diagnosis of these tumors may be difficult because of significant morphologic overlap. However, several recent studies have described morphological features that may help in making these distinctions on the basis of aspiration cytology. 2-8
’
Received September 4, 1991. Accepted January 15, 1992. From the Department of Pathology and Laboratory Medicine and Department o f Oncology, King Faisal Specialist Hospital, and Research Centre, Riyadh, Saudi Arabia. Address reprint requests to Dr. Mohammed Akhtar, Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia. (c)
1992 W I L t > - L I S S . INC
All cases of rhabdomyosarcoma diagnosed by FNAB between January 1980 and December 1990 were identified and of these, a group of cases in which the FNAB diagnosis was further confirmed by histologic examination, were selected for review. Of the 31 cases identified, nine were excluded because the cellular material in the FNAB smears was inadequate for detailed evaluation. Another seven cases were excluded because the material for immunohistochemistry was not available. The remaining 15 cases form the basis of this study (Table I). Fine-needle aspiration biopsies were performed by using 22-25-gauge needles with or without aspiration. Smears were air-dried and stained by Diff-Quik stain. For immunohistochemical studies, a portion of the material was placed in vials containing tissue culture medium (RPMI-I640 Hazelton Cat #50178); 0.1-0.2 ml of the suspension was used to obtain cytospin preparations using 500 RPM for 5 min. These slides were stored in a refrigerator at 4°C for a maximum of 5 days. After fixation in ice-cold acetone for 10 mins, immunoperoxidase staining was performed using ABC technique. The electron microscopy part of the FNAB sample was fixed in 3% glutaDiugnostic Cytoputhology, Vol 8. No 5
465
AKHTAR ET AL. Table I. Clinicopathologic Features of 15 Cases of RMS Case no.
1 2 3 4
5
6 7 8 9 10 I1 12 13 14
15
Location of FNAB
Electron microscopy
I m m unoperoxiduse staining on FNAB
I m m unoperoxidase stuining of titsue
Chest wall Elbow Left groin Left neck Left axilla Rt. upper arm Neck and R t . maxilla Left orbit Gluteal Mediastinurn Abdomen ParaInguinal areas Upper arm and lung
Yes No No Yes Yes Yes No No No Yes No Yes Yes Yes No
Yes
Yes Yes Y es Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Age and sex 9M 13F 19F 9M 1OM 1M 9M 2M 11M
5M 4M 2F 2/12M 15F SM
raldehyde for at least 1 hr and then processed according to the technique described previously. l 4 Tissue biopsy specimens were fixed in 10% buffered formaldehyde solution and embedded in paraffin in a routine fashion. Sections were stained with hematoxylin and eosin stain. For immunohistochemical studies, sections were stained using antibodies against vimentin (DAKO) Desmin (BIOGENICS) and myoglobin (DAKO) using ABC technique.
Results Detailed review of aspiration smears in our cases revealed that the neoplasms were composed of a variable mixture of tumor cells that showed marked variation in size and shape. However, based on the degree of apparent myogenic differentiation, these cells were divided into three categories, namely, early, intermediate, and late rhabdomyoblasts. Early rhabdomyoblasts were round, undifferentiated cells with large, irregular, single nuclei and scanty basophilic cytoplasm (Figs. C-1, C-2). Intermediate rhabdomyoblasts had relatively abundant pale staining variably vacuolated cytoplasm and one or more irregular nucleus with occasional nucleolus (Fig. C-3). Late rhabdomyoblasts contained abundant cytoplasm that stained grayish blue and opaque. These cells varied from round to markedly elongated resembling myotubules (Figs. C-4, C-5).Some of these cells had localized inclusion-like grayish blue areas within the cytoplasm (Fig. C-6). Some of the intermediate and late rhabdomyoblasts were extremely large with markedly pleomorphic nuclei. The relative proportion of various cell types varied considerably from one case to another. Whereas a majority of the tumors contained all three types of cells, in some only two cell types, namely, early and intermediate rhabdomyoblasts, were recognized (Table 11). The tumor cells were loosely arranged without any specific pattern. Occasionally, early rhabdomyoblasts were arranged in small 466
Diagno.rtic Cyioputhology, Voi 8,No 5
No NO
No No No Yes No Yes NO NO
Yes
No Yes NO
tight cell clusters; however, no cell clusters composed of intermediate or late rhabdomyoblasts were encountered (Fig. C-2). Histologic examination of the tumors together with electron microscopy and immunohistochemistry enabled the diagnosis of rhabdomyosarcoma to be made either by identification of rhabdomyoblasts or by characteristic growth pattern such as seen in alveolar rhabdomyosarcoma. These tumors were further subdivided into the following morphologic subtypes: embryonal RMS, 9; monomorphic round cell RMS 4; and alveolar RMS, 2. l 5 Comparison of histopathologic appearance of tumors with cytomorphologic features in aspiration smears indicated a close correlation. Thus embryonal rhabdomyosarcomas, which on histological examination were found to contain large numbers of cells with obvious myogenic differentiation, tended to reveal relatively frequent intermediate and late rhabdomyoblasts (Figs. C-4-C-6). By contrast, alveolar RMS and monomorphic round cell RMS were composed of relatively undifferentiated small round cells (Figs. C-1, C-2). Evidence of myogenic differentiation in these tumors was found only after careful examination of several sections. Corresponding FNAB smears revealed large numbers of early rhabdomyoblasts with variable scattering of intermediate cells and only rare late rhabdomyoblasts (Table 11). Ultrastructurally, early rhabdomyoblasts contained small amount of cytoplasm with large numbers of ribosomes, few mitochondria, and occasional profiles of rough endoplasmic reticulum (Fig. 1). Intermediate rhabdomyoblasts contained relatively abundant cytoplasm rich in organelles such as mitochondria, profiles of rough endoplasmic reticulum and variable numbers of lyosomes and aggregates of glycogen (Figs. 2, 3). Some of these cells in addition revealed small parallel aggregates of thick (myosin) and thin (actin) filaments (Fig. 4). Late rhab-
RHABDOMYOSARCOMA DIAGNOSIS Table 11. Cytologic, Histologic, and Ultrastructural Correlations
Case no.
2 4 7 9 10 11
12 13
I5 I 14 3 5 6 8
Histologic subtype
Immunohistochemical findingv'
Embryonal Embryonal Embryonal Embryonal Embryonal Embryonal Embryonal Embryonal Embryonal Alveolar Alveolar Monomorphic Monomorphic Monomorphic Monomorphic
"V = vimentin, D = desmin, M
V+D+M+ V+D+M+ V+D+MV+D+M+ V+D+M+ V+D+M+ V+D+M+ V+D+MV+D+M+ V+D+MV+D+MV+D+MV+D+MV+D+M+ V+D+M=
in
15 Cases of RMS
Myogen ic diffcrentrotion
Cytologic types of rhabdornyoblasts Early ++i
++ ++ + + + ++ ++ ++++ ++++ +++ ++++ ++++ +++
In term edia te
+ ++ + +
++ ++ ++ ++ ++ + + ++ + + +
Lute t
+ ++ +++ ++ ++ t + f i
ot7
Not Ye\ Not Not Ye\ Not Ye\ Ye\ Not Yes Not Not Not Ye\ Not
EM done
done done done
done present done pre\ent
done
myoglobin.
Fig. 1. Electronmicrograph depicting early rhabdornyoblasts in FNAB from RMS. The tumor cells are undifferentiated with not clear evidence of myogenic differentiation (Uranyl acetate and lead citrate stain, x 12,000). Diagnostic Cytopathology, Vol 8, No 5
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Fig. C-1A
Fig. C-1B
Fig. C-ZA
Fig. C-ZB
Fig. C-3A
Fig. C-3B
Figs. C - 1 4 - 3 . Fig. C-1. A. FNAB smear, featuring early rhabdomyoblasts with large nuclei and relatively scanty cytoplasm (Diff-Quik stain, X 150). B. Histologic section from a monomorphic round cell RMS composed of undifferentiated round cells (Diff-Quik stain, X 150). Fig. C-2. A. Early rhabdomyoblasts forming small clusters in an alveolar RMS (Diff-Quik stain, X 150). B. Histologic section showing a representative area i n alveolar RMS. The tumor cell\ are small and undifferentiated and tend to form alveolar spaces (Hematoxylin & Eosin stain, x 100). Fig. C-3. A. Aspiration smear showing loosely arranged intermediate rhabdomyoblasts (Diff-Quik stain, X 150). B. Higher magnification photomicrograph showing intermediate rhabdomyoblasts with abundant pale staining cytoplasm. Some of the cells have multiple nuclei (Diff-Quik stain, X 250).
Fig. C-4A
Fig. C-4B
Fig. C-5A
Fig. C-5B
Fig. C-6A
Fig. C-6B
Figs. C - 4 4 - 6 Fig. C-4. A. Aspiration cells depicting four late rhabdomyoblasts. The cytoplasm is abundant, grayish blue and uniformly opaque (Diff-Quik stain, X 200). B. Histologic sections from embryonal RMS showing cells with abundant, diffusely eosinophilic cytoplasm indicating myogenic differentiation (Hematoxylin and Eosin stain, x 150). Fig. C-5. A. Aspiration smear showing several elongated late rhabdomyoblasts from embryonal RMS (Diff-Quik stain, X 200). B. Corresponding histologic section showing well-differentiated rhabdornyoblasts (Hematoxylin and Eosin stain, X 150). Fig. C-6. A. A late rhabdomyoblast in aspiration smear containing an elongated inclusion-like area within the cytoplasm (Diff-Quik stain, X 250). B. Histologic section showing several rhabdomyoblasts with eosinophilic inclusion-like areas of myogenic differentiation (Hematoxylin and Eosin stain, x200).
Fig. 2. Electronmicrograph featuring intermediate rhabdomyoblasts. The cytoplasm is relatively abundant with various organelles (Uranyl acetate and lead citrate stain, x 14,000).
Fig. 3. Another intermediate rhabdomyoblast with abundant cytoplasm, which X 5,700).
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IS extremely
rich in organelles (Uranyl acetate and lead citrate stain,
RHABDOMYOSARCOMA DIAGNOSIS
domyoblasts contained cytoplasm similar to that seen in intermediate rhabdomyoblasts. However, these cells in addition contain larger aggregates of thick and thin filaments with varying degrees of sarcomeric organization. These zones of myogenic differentiation may involve small localized areas of the cytoplasm or may virtually replace the entire cytoplasm with virtual exclusion of other organelles (Fig. 5).
Discussion FNAB diagnosis of RMS is primarily dependent upon demonstration of cells that could be clearly identified as rhabdomyoblasts. Late rhabdomyoblasts with their distinctive opaque cytoplasm may be identified with relative certainty. Intermediate rhabdomyoblasts with relatively abundant pale cytoplasm may also be tentatively recognized, but their precise identity may only be established by immunocytochemistry and/or ultrastructural examination. Early rhabdomyoblasts are morphologically undifferentiated cells that closely mimic similar cells in other types of round cell malignancies. These cells are generally without any demonstrable ultrastructural features of myogenic differentiation. Immunocytochemistry, in contrast, is much more likely to provide a positive identification since most of these cells stain positive for vimentin and desmin. The role of electron microscopy in the diagnosis of RMS IS well-established. I6-l8 The material for electron microscopy may be derived from a tissue biopsy or fineneedle aspiration biopsy. The minimum requirement for ultrastructural diagnosis is the demonstration of actin and myosin (thick) filaments or recognition of ribosome-myosin complex. More differentiated cells may show parallel organization of sarcomeres with formation of M-lines and Z bands. Ultrastructural evidence of myogenic differentiation may be easily obtained in FNAB material from embryonal rhabdomyosarcoma; however, it may be difficult in other types of RMS.2Q^2‘Tumors with strong immunohistochemical evidence of rhabdomyomatous differentiation (usually strong desmin positivity) but without ultrastructural features of muscle differentiation represent a difficult diagnostic dilemma. Some authors have characterized such tumors as RMS-like tumors. 2 1 Others argue that failure to demonstrate organized myofilaments does not exclude the possibility of poorly differentiated rhabdomyosarcoma. 2o In two cases ultrastructural evidence of myogenic differentiation could not be found, although tumor cells in both were strongly positive for desmin. One of these had morphological features characteristic of alveolar RMS. Admittedly, this discrepancy may be explained on the basis of sampling error. However, we believe that a more plausible explanation maybe that desmin is expressed within rhabdomyoblasts before any myofilaments can be recognized.
A large number of immunocytochemical markers for diagnosis of RMS have been studied. These include desmin, skeletal muscle specific actin, muscle specific actin, myoglobin, myosin, troponin, creatine kinase, Z-band protein, and titen. 14*21-24However, the most widely used marker is desmin, which is an intermediate filament protein located in close association with Z-band material. The sensitivity and specificity of desmin in the diagnosis of RMS have been the subject of numerous studies. It is now generally agreed that Desmin is an extremely sensitive and a fairly reliable marker for smooth and striated muscle differentiation. 23-25 Smooth muscle neoplasms are rare in children and therefore do not cause any significant diagnostic difficulty with rhabdomyosarcoma. Other conditions in which desmin positivity may occasionally be seen include Wilms’ tumor, malignant fibrous histiocytoma, glial tumors, malignant Schwannoma, malignant melanoma, malignant mesothelioma, fibromatosis, renal rhabdoid tumor, myositis ossificans, and embryonal sarcoma of liver. 22-23 Most of these lesions, however, rarely pose diagnostic difficulty with rhabdomyosarcorna in children. Nevertheless, we believe that full spectrum of desmin reactivity in various benign and malignant neoplasms is yet to be determined. Consequently, it is advisable to confirm cytologic and histologic diagnosis of rhabdomyosarcoma by immunohistochemistry as well as electronmicroscopy in as many cases as possible. Correlation of cytologic, histologic, and ultrastructural findings in our study indicates that early, intermediate, and late rhabdomyoblasts reflect various stages in the differentiation of tumor cells. Early rhabdomyoblasts are primitive cells in which there is no morphologic evidence of myogenic differentiation. However, on immunocytochemistry, most of these cells stain with desmin indicating a commitment to such a differentiation. Ultrastructurally, these cells are devoid of actin and myosin filaments. The intermediate myoblasts are comparatively more differentiated but still do not reveal any definite evidence of myogenic differentiation at light microscopic level. Ultrastructurally, however, a few small cytoplasmic aggregates of thin and thick filaments may be seen, although they are not arranged to form distinct bands. Late rhabdomyoblasts are cells that show obvious myogenic differentiation at light microscopy in the form of dense, opaque areas within the cytoplasm. These areas involve variable portion of the cytoplasm. Ultrastructural appearance of these areas corresponds to the presence of large bundles of thick and thin filaments with sarcomeric arrangements. When myogenic differentiation is limited to a small localized area within the cytoplasm, it may appear as an inclusionlike area. These inclusions are also composed of thick and thin filaments with Z-band formation. In a recent study rhabdoid-like inclusions have been described in some of the rhabdomyosarcomas. 27 These are ultrastructurally Diagnostic Cyropathology, Vol 8, NO 5
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Fig. 4. Higher power electron micrograph revealing small bundles of thick and thin filaments in an intermediate rhabdomyoblart (Uranyl acetate and lead citrate stain, x 25,000).
Fig. 5. A-C. Late rhabdomyoblasts depicting various patterns of myogeriic differentiation (Uranyl acetate and lead citrate stain, A : X 14,000; B: x 12,000;c: x Is,oao).
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composed of aggregates of intermediate filaments and are thus different from the myogenic inclusions seen in our study. Rhabdomyosarcoma is not the only neoplasm in which rhabdomyoblasts may be present. Focal rhabdomyoblastic differentiation occurs in a variety of malignant neoplasms such as malignant mixed tumor of the uterus and ovary, carcinosarcoma of breast, teratoma, pulmonary blastoma, hepatoblastoma, and nephroblastoma. In addition, rhabdomyoblastic differentiation is also a feature of various neuroectodermal neoplasms notably malignant Schwannoma (malignant Triton tumor), ectomesenchymoma and medullo-epithelioma. ** Fine-needle aspiration biopsy smears from these neoplasms may be extremely difficult to interpret. Evaluation of clinical features may help to exclude some of these tumors. Precise FNAB diagnosis, however, will depend upon recognition of the cellular patterns of the parent tumor. Fortunately, with the exception of Wilms’ tumor, these neoplasms are rare. We have encountered rhabdomyoblasts in the aspiration smears from some of the Wilms’ tumors that we have studied. In these cases, recognition of the typical cellular components namely, blastemal, tubular, and stromal patterns served to establish FNAB diagnosis of Wilms’ tumor with rhabdomyomatous elements. In summary, FNAB findings in a series of 15 cases of RMS are described and correlated with histologic and ultrastructural features. It is suggested that embryonal rhabdomyosarcoma with many late and intermediate rhabdomyoblasts may easily be diagnosed on FNAB. Undifferentiated RMS, in contrast, would generally require immunohistochemistry and/or electron microscopy.
’
References 1. Triche HM, Askin FB, Kissane JM. Neuroblastoma Ewing’s sarcoma and the differential diagnosis of small round blue cell tumors.
2.
3.
4. 5.
6.
7.
In: Finegold M ed. Pathology of neoplasia in children and adolescents. Major Problems in Pathology. Philadelphia: WB Saunders, 1986;18:14S-95. Akhtar M, Ali MA, Sabbah R, Bakry M, Nash JE. Fine needle aspiration biopsy diagnosis of round cell malignant tumors of childhood: a combined light and electron microscopic approach. Cancer 1 985:5 5: 1805- 17. Akhtar M, Ali MA, Sabbah R. Aspiration cytology of Ewing’s sarcoma: light and electron microscopic correlations. Cancer 1985; 56:2051-60. Miller JR, Bottles K , Abele JS, Becksted JH. Neuroblastoma diagnosed by fine needle aspiration biopsy. Acta Cytol 1985;29:461-8. Akhtar M, Ali MA, Sabbah R, Bakry M, Sackey K , Nash EJ. Aspiration cytology of neuroblastoma: light and electron microscopic correlations. Cancer 1986;57:797-803. Silverman JF, Dabbs DJ. Ganick DJ, Holbrook CT, Geisinger KR. Fine needle aspiration cytology of neuroblastoma including peripheral neuroectodermal tumor with immunohistochemical and ultrastructural confirmation. Acta Cytol 1988;32:367-76. Valkov L, Boijikin B. Fine needle aspiration biopsy of abdominal and retroperitoneal tumors in infants and children. Diagn Cytopatho1 1987:3:129-33.
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8 Akhtar M, Ali MA, Sackey K, Sabbah R, Burgess A. Aspiration cytology of Wilms’ tumor: Correlation of cytologic and histologic features. Diagn Cytopathol 1989;5:269-74. 9 Mariarty AT, Nelson WA, McGahey B. Fine needle aspiration of rhabdomyosarcoma of heart: light and electron microscopic findings and histologic correlation. Acta Cytol 1990;34. 10 Pettinato G, Swanson PE, Insabato L, Chiara AD, Wick MR. Undifferentiated small round-cell tumors of childhood: the immunohistochemical demonstration in fine needle aspirates. Diagn Cytopathol 1989;s:194-9. 11 Seidal T, Walaas L, Kindblom LG, Angervall L. Cytology of embryonal rhabdomyosarcoma. A cytologic, light microscopic, electron microscopic and immunohistobhemical study of seven cases. Diagn Cytopathol 1988;4:292-9. 12 Torres V, Ferrer R. Cytology of fine-needle aspiration biopsy of primary breast rhabdomyosarcoma in an adolescent girl. Acta Cytol 1985;29:43M. 13 de-Jong AS, Van-Kessel-Van Vark M, Van-Heerde P. Fine needle aspiration biopsy diagnosis of rhabdomyosarcoma: an immunohistochemical study. Acta Cytol 1987;31:573-7. 14 Akhtar M, Bakry M, Nash EJ. An improved technique for processing aspiration biopsy for electron microscopy. Am J Clin Pathol 1986;85:57-60. 15 Dodd S, Malone M, McCulloch W. Rhabdomyosarcoma in children: a histologic and immunohistochemical study of 59 cases. J Pathol 1989;158: 13-18. 16 Erlandson RA. The ultrastructural distinction between rhabdomyosarcoma and other undifferentiated sarcomas. Ultract Pathol 1987;11 183-101. 17 Siedal T, Kindblom LG. The ultrastructure of alveolar and embryonal rhabdomyosarcoma: a correlative light and electron microscopic study of 17 cases. Acta Pathol Microbiol Immunol Scand Sect 1984; 92:23148. 18 Mierau GW, Favara BE. Rhabdomyosarcoma in children: ultrastructural study of 31 cases. Cancer 1980;46:203540. 19 Ghadially FN. Diagnostic electron microscopy of tumors, 2nd ed. London: Butterworth, 1985; 186-205. 20 Seidal T, Kindblom LG, Angervall L. Alveolar and poorly differentiated rhabdomyosarcoma: a clinicopathologic light-microzcopic ultrastructural and immunohistochemical analysis. APMIS 1988;96: 825-38. 21 Miettinen M, Rapola J. Immunohistochemical spectrum of rhabdomyosarcoma and rhabdomyosarcoma-like tumors: expression of cytokeratin and 68-KD neurofilament protein. Am J Surg Pathol 1989; 13:120-32. 22 Molenaar WM, Oosterhius JW, Oosterhius AM, Ramaeker FCS. Mesenchymal and muscle specific intermediate filaments (vimentin and desmin) in relation to differentiation in childhood rhabdomyosarcoma. Human Pathol 1985;16:83843. 23 Truong LD, Rangdaeng S, Cagle P, Ro TY, Hawkins H, Font RL. The diagnostic utility of desmin: study of 584 cases and review of literatures. Am J Clin Pathol 1990;93:305-14. 24 Leader M, Patel J, Collins M, Henry K. Myoglobin: an evaluation of its role as marker of rhabdomyosarcoma. Br J Cancer 1989;59: 106-9. 25 Ordonez NG. Antidesmin antibodies (Editorial): their use in diagnostic pathology. Am J Clin Pathol 1990;93:43&1. 26 Parhan DM, Webber B, Holt H, Williams WK, Maurer M. Inimunohistochemical study of childhood rhabdomyosarcoma and related neoplasms: results of an intergroup rhabdomyosarcoma study project. Cancer 1991:67;3072-80. 27 Kodet R, Newton WA, Hamoudi AB, Asmar L. Rhabdomyosarcomas with intermediate filament inclusions and features of rhabdoid tumors: light microscopic and immunohistochemical study. Am J Surg Pathol 1991;15:257-67. 28 Enzinger FM, Wiess SW. Rhabdomyosarcoma. In: Enzinger FM and Wiess SW, eds. Soft tissue tumors. St. Louis: C.V. Mosby, 1988:448-88.
