Expression of Developmentally Regulated Muscle Proteins in Rhabdomyosarcomas

Transcription

1 Amneican jornal of Pathology, Vol. 45. No. 4, October 994 Coyright C) American Society for Investigative Patbology Exression of Develomentally Regulated Muscle Proteins in Rhabdomyosarcomas Liliane C.D. Wijnaendts,* Johannes C. van der Linden,* Adriaan J.M. van Unnik,t Jan F.M. Delemarre,* J. Patrick Barbet, Gillian S. Butler-Browne, and Chris J.L.M. Meijer* From the Deartment of Pathology, * Free University of Amsterdam, Amsterdam, 7he Netherlands; the Deartment of Pathology,t Groot Zieken Gasthuis of Den Bosch; the Deartment of Pathology,t Antoni van Leeuwenhoek Ziekenhuis, Netherlands Cancer Institute ofamsterdam, Amsterdam, The Netherlands; the Deartment of Pathology, Hoital Saint Vincent de Paul of Paris, Pads, France; and the CNRS URA 448, Pars, France Human skeletal muscle differentiation and maturationfollows a recise sequence ofevents. To investigate whether and to what extent rhabdomyosarcoma (RMS) cells folow a comarable sequence, 9fresh frozen secimens of RMS (4 rimary and 5 relases) were immunostained with antibodies directed against develomentaly regulated myosin heavy chains (MHC), ie, fetal, fast, and slow MHC, in addition to desmin and vimentin. Four distinct atterns of exression were observed. I) RMS ceus exressing exclusively vimentin and desmin (n = 7), II) in addition to exression ofvimentin and desmin, a minority ofneolastic ceus were immunoreactive withfetal MHC (n = 6), III) in addition to attern II, fast MHC was exressed (n = 7), and IV) RMS cells simultaneously exressing vimentin, desmin, fetal, fast, and slow MHC (n = 9). Accordingly, the roortion ofthe MHC immunoreactive RMS cells increased gradually along with thefouratterns of exression evolving from less than 5% u to 75%forfetal MHC, from less than 5% u to 5% forfast MHC, and u to 5%for slow MHC in the last category. Vimentin and desmin were coexressed by almost all RMS cells. Double immunostaining revealed that comarable with the myogenic cells in the develoing fetal skeletal muscle, exression offetal MHC could be demonstrated in the same neolastic cells either in conjunction with fast or slow MHC. In contrast, only in RMS, slow MHC exression in conjunction withfast MHC could be observed in the neolastic ceus. Neither the shae or size ofneolastic RMS cells, nor the histoathological tyes, nor tumor localization were related to the exression attern of develomentaly regulated MHC (fetal, fast, and slow MHC). These results confirm the commitment ofthe RMS cells to the myogenicathway and demonstrate a restricted and aberrant differentiation attern of the neolastic ceus in RMS comared with normal myogenesis, indeendent of histoathological tyes ofrms. (Am J Pathol 994, 45:895-9) Rhabdomyosarcoma (RMS) is a highly malignant neolasm of myogenic origin accounting for a major roortion of soft tissue sarcomas in infants and children.' The diagnosis is based on the resence of rhabdomyoblasts, resembling striated muscle cells at initial stages of differentiation. RMS often dislay a wide morhological sectrum ranging from oorly differentiated cells to well-differentiated rhabdomyoblasts, which characteristically dislay registered or hahazardly organized sarcomeric comonents and cross-striations. Poorly differentiated samles are sometimes difficult to distinguish from other round cell tumors of soft tissues such as neuroblastoma, Ewing sarcoma, and rimitive neuroectodermal tumors. In these cases, markers of early myogenic differentiation are essential in the differential diagnostic rocedure. In normal skeletal muscle, the develomental rogram occurs through a discrete series of structural and functional events accomanied by changes in the exression of secific tyes of intermediate filament roteins (vimentin and desmin)3 and relacement of molecular variants of some contractile ro- Suorted in art by the Ligue Nationale Francaise center le Cancer and the Association Francaise contre les Myoathies. Acceted for ublication July 7, 994. Address rerint requests to Dr. Liliane C.D. Wijnaendts, Deartment of Pathology, Free University of Amsterdam, Boelelaan 7, 7 MB Amsterdam, The Netherlands. 895

2 896 Wijnaendts et al AJP October 994, Vol. 45, No. 4 teins.4 Vimentin is redominantly synthesized by the relicative resumtive myoblasts. On fusion, myotubes synthesize high levels of desmin. As myotubes mature, vimentin synthesis is gradually decreased till the 3th week of gestation while roduction of desmin is maintained.35 Myosin, the major comonent of the contractile aaratus, is comosed of two heavy chains and four light chains.6 The myosin heavy chain (MHC) has multile isoforms including the embryonic, fetal, fast, and slow MHC. The relacement of the develomental MHC isoforms (embryonic and fetal MHC) by the adult MHC isoforms (fast and slow) is comleted just before or just after birth. In a recent study by Barbet et al,5 two generations of myotubes were defined during the fetal develoment of the human quadrices, each based on a characteristic exression of develomentally regulated MHC isoforms. The first generation of muscle fibers formed between 8 and weeks of gestation all exress slow MHC in addition to the develomental isoforms (embryonic and fetal MHCs). Final maturation of the first generation fibers is reached in 8 weeks when they eliminate the develomental isoforms, resulting in sole exression of slow MHC. Between and 8 weeks, fibers of the second generation exressing the develomental isoforms and fast MHC form rogressively and asynchronously around the rimary fibers. In weeks the second generation fibers undergo a rocess of maturation and differentiation when they eliminate the develomental isoforms and exress either slow or fast MHC. The aim of this study was to evaluate whether and to what extent RMS cells follow the same sequence of events involved in differentiation and maturation of normal myogenic cells. Therefore, a anel of monoclonal antibodies directed against develomentally regulated MHC, vimentin, and desmin was used. Furthermore, the relationshi between the different immunohistochemical atterns of RMS and histoathological tyes was investigated. Materials and Methods Patients and Control Material Clinical data and 9 frozen secimens from atients, diagnosed for RMS at the Free University Hosital of Amsterdam and the Academic Hosital of Groningen between 983 and 99, were selected for this study. In 4 atients, rimary tumors and eventual ostchemotheray/radiotheray relased tumors (n = 5) were available. In the other 8 atients, (multile) relases were available. The mean age of the atients at the time of diagnosis was 3 years. There were male and female atients. The anatomic sites of the rimary tumors included head and neck (n = 9), urogenital and elvis (n = 6), abdomen (n = 3), extremities (n = ), and thorax/trunk (n = ). Frozen tissue sections of fetal (gestational age ranging from 9 to weeks) and adult skeletal muscle were used as controls. Histoathological Characterization Hematoxylin and eosin-stained slides of the corresonding formalin-fixed, araffin-embedded tissue of rimary RMS were reviewed. A anel of immunohistochemistry including muscle actins, leukocyte common antigen, S-, neuron-secific enolase, and cytokeratins was used to confirm the diagnosis. Tumors were classified, according to reviously described criteria,9 into embryonal RMS (n = ), alveolar/solid alveolar RMS (n = 7), botryoid RMS (n = ), sindle cell RMS (n = ), and RMS not otherwise secified (n = ). Immunohistochemistry Immunohistochemistry was conducted on 5- frozen sections using monoclonal antibodies secific for fetal, slow, and fast MHCs, vimentin (clone V9; Dakoatts and Monosan), and desmin (D33; Monosan). Sections were incubated for hour with the rimary antibody and secific antibody binding was revealed by the avidin-biotin-eroxidase comlex (ABC) (Dakoatts). Co-exression of MHC isoforms in the same RMS cells was analyzed using a double immunostaining technique that combined the ABC and alkaline hoshatase/monoclonal antialkaline hoshatase (APAAP) method (Dakoatts). Fast MHC was made visible by the brown color of diaminobenzidine and slow MHC by the blue color of Fast Blue BB. Several controls were included in each staining series: ) monostaining with each of the monoclonal antibodies with the ABC method and ) double staining sequences in which one (first or second rimary antibody) or two (first and second rimary antibodies) stes were changed into hoshate-buffered saline. Evaluation of immunostaining was erformed by two athologists at a x4 magnification. It was based on the ercentage (from to %) of ositive

3 Myosin Heavy Chain Isoforms in Rhabdomyosarcoma 897 AJP October 994, Vol. 45, No. 4 Table. Immunobistochemical Exression Patterns and Mean Percentage ofimmunoreactive Neolastic Cells of 9 RMS Pattern of Number Fetal Fast Slow Exression of RMS Vimentin Desmin MHC MHC MHC III IV , No ositive neolastic cells; +, mean ercentage immunoreactive neolastic cells s5%; +, mean >5% and -5%; 3+, mean >5% and -75%; 4+, mean >75%. Double staining rocedures revealed co-exression in the same RMS cells of fetal and fast MHC (II-IV), fetal and slow MHC (IV), and fast and slow MHC (IV). neolastic cells in the total tumor surface; in all cases, a minimum of high ower fields were counted. The mean reresented the roortion of immunoreacting cells within a given grou of tumors. Tumors were classified as + when the mean ercentage of immunoreacting cells was <5%, + when the mean was >5% and <5%, 3+ when the mean was >5% and.75%, and 4+ when the mean was >75%. I i If i Ii 4 a...~~~~~~~~~~~~~~~.. Ltf,,7-.;,.S: nm&. Figure. Enmbryonal RMS stainedfor vlirnentin (A), desmini (B), fetal MHC (C), fast MHC (D), and slou MHC (E). Almost all neolastic cells stained for vimentin and desmin. whereas 4,, and % qf neolastic cells stainedforfetal, fast. and slowi MHCI resectively (ABCfinal magnification x36).

4 898 Wijnaendts et al AJP October 994, Vol. 45, No. 4 Results In the 9 secimens of RMS, four distinct atterns of exression were observed (Table ). At first (attern ), the RMS cells only exressed vimentin and desmin (n = 7). Second (attern ), in addition to vimentin and desmin, a minority of neolastic cells was immunoreactive with fetal MHC (n = 6). According to the third attern of exression (attern ), neolastic cells also exressed fast MHC (n = 7), whereas in the last category (attern IV), RMS cells simultaneously exressed vimentin, desmin, fetal, fast, and slow MHC (n = 9) (Figure ). Accordingly, the roortion of RMS cells exressing develomentally regulated MHC increased gradually along with the four atterns of exression evolving from + (-<5%) to 3+ (<5% and.75%) for fetal MHC, from + to + (>5% and -<5%) for fast MHC, and reaching + for slow MHC in the last category. Vimentin and desmin were both exressed by almost all RMS cells; soradically, few large neolastic cells did not exress vimentin (Figure A, B). Furthermore, both the number of immunoreactive tumors and the roortion of immunoreactive RMS cells for the three MHC isoforms was shown to be consistently higher in relased RMS when comared with rimary RMS (Table ). With resect to the size and shae of the neolastic cells immunoreactive with the MHC isoforms, no characteristic attern of MHC isoform exression was found: all three MHC isoforms were indifferently localized in small round tumor cells and in larger round and elongated cells. When the histoathological tyes were regarded searately, no clear association was seen between RMS tyes and immunohistochemical atterns of MHC exression (Table ). Similarly, localization of the tumors was not related to a secific immunohistochemical rofile. Table. Exression Pattern of Histoathological RMS Tye for Fetal, Fast, and Slow MHC RMS Tye Embryonal RMS Alveolar RMS Botryoid RMS Sindle RMS RMS NOS Total r r r Fetal Fast Slow n MHC MHC MHC (+) (+) (+) 9 (±) 3 (+) 6 (+), Primary RMS; r, relased RMS; ( ), mean ercentage immunoreactive RMS cells; +, mean ercentage s5%; +, mean ercentage >5% and s5%. Double staining rocedures revealed co-exression in the same RMS cells of fetal and fast MHC, fetal and fast MHC, fetal and slow MHC, and fast and slow MHC. Co-exression of MHC isoforms in the same RMS cells was analyzed using double stainings. The majority of the cells were exclusively reactive with fetal MHC. In some tumor cells fetal MHC immunoreactivity was associated with simultaneous exression of fast or slow MHC. Conversely, a few neolastic cells were found to be redominantly or exclusively ositive for fast or slow MHC. An interesting finding emerged when double staining rocedures were erformed with RMS co-exressing slow and fast MHC. A minority of rimarily large tumor cells aeared to contain both fast and slow MHC (Figure, A). As a control, a anel of fetal and adult skeletal human muscle was immunostained and showed similar results to those of Barbet et al.5 The results obtained from double staining rocedures were comarable to those of single immunostaining of consecutive slides5 and confirmed that fast and slow MHC isoforms were not co-exressed in the same myotubes/fibers during normal skeletal muscle develoment of the quadrices. Discussion Human skeletal muscle differentiation and maturation after a recise sequence of events have been reviously immunohistochemically characterized using antibodies directed against vimentin, desmin, and the develomentally regulated MHC isoforms.58 In this study, the immunohistochemical rofile of 9 RMS was shown to evolve according to four distinct atterns (atterns I-IV) of MHC isoform exression (Table ). Comarison of these atterns in RMS with the develoing human fetal quadrices suggests that as a grou RMS resemble a relatively restricted segment of fetal muscle develoment. This segment is bounded on one end by the commitment of cells to the myogenic athway, ie, immunoreactivity for vimentin, desmin (attern ), and eventually scant immunoreactivity for fetal MHC (attern ), and on the other by the earliest overt stages of myogenesis, ie, increased immunoreactivity for fetal MHC and additional immunoreactivity for fast MHC (attern ) and slow MHC (attern IV), whereas immunoreactivity for vimentin remained resent in almost all neolastic cells (Table ). Based on the fact that neolastic cells from RMS relases reresent more differentiated stages of RMS caused by treatment with chemotheray," the higher immunoreactivity for adult MHCs (fast and slow) in relased RMS illustrates the differentiation otential of the RMS cells. Indeed, when comared with rimary RMS, a higher number of immunoreactive tumors and a higher roortion of immunoreactive tumor cells

5 Myosin Heavy Chain Isoforms in Rhabdomyosarcoma 899 AJP October 994, Vol. 45, No. 4 Figure. Embryonal RMS (A) and skeletal muscle tissue from the quadrices of a human fetus of 3 weeks gestation (B) double stained for fast MHC (brown color) and slow MHC (blue color) (ABC and APAAPfinal magnification x36 [A] and x9 [B]). were found for both fast and slow MHC in relased RMS (Table ). During the fetal skeletal muscle develoment, various stages of differentiation and maturation may be simultaneously resent. This exlains the co-existence of cells exressing exclusively fetal, fast, or slow MHC and cells co-exressing fetal and adult MHC. Such co-exressions in the same RMS cell have reviously been reorted. 4-6 On the other hand, in our double staining essays (Figure, B) and the results of Barbet et al,5 co-exression in the same myotubes/fibers of fast and slow MHC was not found. Therefore, the resence of double staining for fast and slow MHC in the same mature tumor cells resumably reflects an aberrant differentiation within these tumor cells. The fact that RMS cells, unlike normal myoblasts in vivo, grow indeendently of motor innervation and motor activity might contribute to the develoment of this aberrant henotye. This result is very similar to that seen in human satellite cells grown in culture that are able to co-exress both adult fast and slow MHCs in addition to embryonic and fetal MHCs.7 Moreover, a arallel can be drawn with T cell lymhomas in which aberrant henotyic exression, such as simultaneous exression of B and T cell markers (lineage romiscuity), can be found. 8 The exression of vimentin and desmin in almost all tumor cells was consistent with the myogenic nature of these tumors. It has reviously been reorted that on the analogy of normal myogenesis, develoment of rimitive tumor cells to morhologically recognizable myogenic tumor cells was accomanied by an increase in desmin ositivity and a decrease in vimentin ositivity.9, In our series, excet for the few large vimentin-negative RMS cells, vimentin and desmin immunoreactivity was comarable. The fact that unlike this study immunohistochemical staining was reviously erformed on formalin-fixed material oint to a ossible fixation factor as the cause of this

6 9 Wijnaendts et al AJP October 994, Vol. 45, No. 4 discreancy. The observed differences in vimentin reactivity would, therefore, reflect quantitative differences. The focus of our study was to have analyzed consistently the exression of MHC isoforms, vimentin, and desmin in a grou of well-documented fresh frozen RMS using monoclonal antibodies. Excet for the study of Barbet et al in which fresh frozen material was used, revious evaluations of the MHC exression attern in RMS were only erformed on formalinfixed, araffin-embedded tissue.6,,3 Furthermore, not all MHC isoforms were consistently evaluated in each study. Now that these four MHC exression atterns have been recognized, it seems that taken together revious investigations suort the existence of these MHC exression atterns (Table ). Barbet et ai studying one RMS found a cumulative sequence of fetal to fast and slow MHC. Eusebi et al6, reorted reactivity for fetal MHC in 8% of the RMS, for fast in 67%, and for slow MHC in 38 to 4%. De Jong et a3 and Carter et a described reactivity for fast MHC in 87 and 55% of the RMS, resectively, whereas all RMS were unreactive with slow MHC in the former study. The histoathological RMS tyes showed a great deal of overla in their differentiation and maturation state, as defined by the exression of develomentally regulated MHC isoforms, vimentin, and desmin (Table ). Therefore, these results suggest that histoathological variance within RMS cannot be exlained on the basis of malignant transformation of cells at different stages of the myogenic athway, as was reviously suggested.4 Similar results were reorted by Tonin et a5 in a recent study in which the genetic rofile of eight alveolar and eight embryonal RMS was determined using a set of genes referentially exressed in normal muscle tissue, ie, myogenic regulatory genes (MyoD, myogenin, MRF4, and Myf5), actins, myosins, and creatine kinase. In conclusion, our results confirm the commitment of the RMS cells to the myogenic athway and demonstrate a restricted and aberrant differentiation attern of the neolastic cells in RMS comared with normal in vivo myogenesis, indeendent of histoathological tyes. Acknowledgments We thank Dr. W. M. Molenaar and W. A. Kams from the Academic Hosital of Groningen for roviding tissue materials and clinical information concerning the atients. Mrs. T. M. Tadema and Mrs. T. Uiterwijk are thanked for exert technical assistance. References. Enzinger FM, Weiss SW: Soft Tissue Tumors. Edited by Stamathis G, Elliott KC, Gregory PL. St. Louis, Mosby- Year Book, 988, Ruymann FB, Grufferman S: Introduction and Eidemiology of Soft Tissue Sarcomas. Edited by Maurer HM, Ruymann FB, Pochedly C. Boca Raton, FL, CRC Press Inc, 99, Lilienbaum A, Li Z, Butler-Browne GS, Bolmont C, Grimaud JA, Paulin D: Human desmin gene: utilization as a marker of human muscle differentiation. Cell Mol Biol 988, 34: Calan Al, Fiszman MY, Eenberger HM: Molecular and cell isoforms during develoment. Science 983, : Barbet JP, Thornell LE, Butler-Browne GS: Immunohistochemical characterization of two generations of fibers during the develoment of the human quadrices muscle. Mechanisms Dev 99, 35:3-6. Lowey S: Myology. Edited by Engel AG, Banker BQ. New York, McGraw-Hill, 986, Pette D, Staron RS: Cellular and molecular diversities of mammalian skeletal muscle fibers. Rev Physiol Biochem Pharmacol 99, 6: Butler-Browne GS, Barbet JP, Thornell LE: Myosin heavy and light chain exression during human skeletal muscle develoment and recocious muscle maturation induced by thyroid hormone. Anat Embryol 99, 8: Wijnaendts LCD, van der Linden JC, van Unnik AJM, Delemarre JFM, Voute PA, Meijer CJLM: Histoathologic classification of childhood rhabdomyosarcomas: relationshi with clinical arameters and rognosis. Hum Pathol (in ress). Ecob-Price M, Hill M, Brown W: Immunocytochemical demonstration of myosin heavy chain exression in human muscle. J Neurol Sci 989, 9:7-78. Hsu SM, Raine L, Fanger H: The use of avidin-biotineroxidase comlex (ABC) in immunoeroxidase techniques: a comarison between ABC and unlabeled antibody (PAP) rocedures. J Histochem Cytochem 98, 9: Mullink H, Henzen-Logmans SC, Alons-van Kordelaar JJM, Tadema TM, Meijer CJLM: Simultaneous immunoenzyme staining of vimentin and cytokeratins with monoclonal antibodies as an aid in the differential diagnosis of malignant mesothelioma from ulmonary adenocarcinoma. Virchows Arch Cell Pathol 986, 5: Molenaar WM, Oosterhuis JW, Kams WA: Cytologic "differentiation" in childhood rhabdomyosarcomas following olychemotheray. Hum Pathol 984, 5: Azzarello G, Sartore S, Saggin I, Gorza L, D'Andrea E, Chieco-Bianchi L, Schiaffino S: Myosin isoform exression in rat rhabdomyosarcoma induced by Moloney murine sarcoma virus. J Cancer Res Clin Oncol 987, 3:47-49

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