sarcoma, and seven malignant fibrous histiocytomas .... quently treated with RNAse (Worthington no. ..... Iwasaki H, Kikuchi H, Takii M, Enjoji R: Benign and.
American Journal of Pathology, Vol. 131, No. 3, June 1988 Copyright © American Association of Pathologists
Characterization ofHuman Soft Tissue Sarcomas in Nude Mice Evidence for Histogenic Properties ofMalignant Fibrous Histiocytomas
P. J. M. ROHOLL, PhD, D. H. RUTGERS, PhD, L. H. P. M. RADEMAKERS, PhD, R. A. DE WEGER, PhD,J. R.J. ELBERS, MD, andJ. A. M. VAN UNNIK, PhD, MD
From the Institute of Pathology, University of Utrecht, The Netherlands; the Department ofRadiotherapy, University Hospital Utrecht, The Netherlands; and the Department ofPathology, St. Antonius Hospital, Nieuwegein, The Netherlands
Twenty-two human sarcomas were grafted subcutaneously into nude mice. Twelve tumors grew successfully. Nine of these 12 tumors had an aneuploid DNA content, whereas only 1 of 10 nonsuccessful tumors was aneuploid. The 12 sarcomas included two leiomyosarcomas, two malignant schwannomas, one synovial sarcoma, and seven malignant fibrous histiocytomas (MFHs). With light and electron microscopic and immunolabeling studies the original and xenografted tumors (the latter for at least two generations) were histopathologically compared. The xenografted leiomosarcomas showed ultrastructurally a more pronounced leiomyodifferentiation, and one of the malignant schwannomas a more pronounced schwannian differentiation. The second malignant schwannoma and the
synovial sarcoma, however, remained unchanged. Five storiform pleomorphic MFHs expressed features that were not observed in the original tumors. Tumor cells of three of these xenografted sarcomas showed leiomyogenic differentiation (filamentous densities, pinocytotic vescicles, and desmin immunoreactivity), whereas cells of the two others demonstrated schwannian differentiation (long cytoplasmic processes, basal lamina). A xenografted myxoid MFH and a pleomorphic MFH gave rise to pleomorphic sarcomas composed of undifferentiated cells. It appeared that under transplantation conditions tumor cells of storiform pleomorphic MFH can differentiate into various directions. (AmJ Pathol 1988, 131:559-568)
CLASSIFICATION of tumors is mainly based on histogenetic grounds, and the ultimate diagnosis is preferentially based on the most differentiated cell type forms in the tumor. Enzinger and Weiss' classified the soft tissue tumors (STTs) according to this principle. This classification was made with light microscopic (LM) observations, later extended with electron microscopic (EM) characterization, but recently the classification could be further improved with applications of immunolabeling techniques.2'3 Within the group of STT the diagnosis of malignant fibrous histiocytoma (MFH) is made mainly on basis of lack of differentiation features; and therefore, it is not surprising that the tissue or cell of origin remains unknown.4'5 Even a characteristic marker pattern cannot be indicated; different markers many ofwhich are considered as indicators for different cell types can be found with im-
munolabeling on paraffin or on frozen sections.6-2 Also, in vitro studies have indicated different cell types of origin, namely, cells with or without the immunologic characteristics of histiocytes. 13-15 Cell lines established from two different MFHs were found to be different; one consisted of a primitive mesenchymal cell and the other one of a fibroblastlike cell. 16 Another cell biologic approach to this histogenetic Supported in part by a grant from The Netherlands Cancer Foundation, Koningin Wilhelmina Fonds (Grant UUKC 82-1 1), and by a grant from Maurits and Anna de Kock Foundation (Amsterdam). Accepted for publication February 1, 1988. Address reprint requests to P. J. M. Roholl, PhD, Institute of Pathology, Department of Histochemistry and Electronmicroscopy, Pasteurstraat 2, 351 1 HX Utrecht, The Netherlands.
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problem is grafting of human tumors into nude mice. 17,18 Grafting of sarcomas offers the opportunity to study the morphologic and immunophenotypic variations of STTs. Hajdu et al investigated the LM histopathology under different grafting conditions. 8 In continuation of an earlier study,'6 we report here the results of 12 xenografted sarcomas, seven ofwhich are MFHs. We have investigated the LM, EM, and immunolabeling characteristics for at least two in vivo passages.
Materials and Methods Tumors STT were freshly obtained from surgical specimen. Under sterile conditions these fragments were freed from necrotic and connective tissue and further divided for multiparametric studies. The histopathologic diagnosis was based on LM aided by EM and immunochemical studies. LM was performed on routinely fixed, processed, and stained sections. Oil red "O" stains were applied on frozen sections of fixed tissues. For EM studies the tissues were fixed in diluted Karnovsky fxative, osmicated, and embedded in Epon 812 according to routine procedures.'9 The immunoperoxidase studies paraffin and frozen sections were used. The avidin-biotin complex (ABC) peroxidase was applied on paraffin sections,20 whereas on frozen sections the indirect immunoperoxidase labeling technique was used.'0 DNA flow cytometry was performed on single cell suspensions.2' Briefly, cells were obtained by enzymatic digestion of the tissue fragments, washed in serum-free medium, resuspended in medium, fixed in a methanol:glacial acetic acid mixture (3: 1), and subsequently treated with RNAse (Worthington no. 5680). After staining with ethidium bromide the DNA content of the cells was analyzed according to Van Der Linden.22 In order to standardize machine setting, murine thymocytes were analyzed prior to each measurement. For transplantation studies inbred nu/nu mice of BALB/c genetic background were used at the age of approximately 3 months. Fragments of tumor (approximately 10 x 2 x 2 mm in size) were transplanted subcutaneously onto the flank. Mice were kept under special pathogen-free conditions, and growth of the grafts was determined by measuring its length (1) and breadth (b) with Vernier calipers under sterile conditions. On basis of these measurements the tumor volume (v) was calculated (v = 0.4 x 1 x b2) in order to determine the growth velocity. For each generation five to ten mice were grafted with tumor tissue. A new
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generation was obtained by transplantation of xenografted tumor tissue. Metastases were never found. The take rate ofthe successful transplantations varied between 40% and 100%. Reagents and Antisera The following heterogenous antisera have been used: rabbit anticow S-100 protein, antineuron-specific enolase (NSE) and antihuman myelin basic protein (MBP) from DAKO-patts (Copenhagen, Denmark), rabbit anti-Factor VIII related antigen from Behringwerke (Marburg, Germany), rabbit antivimentin, antidesmin, antikeratin, and antiglial fibrillar acidic protein (GFAP) from Eurodiagnostics (Apeldoorn, The Netherlands), rabbit antimouse laminin from Bethesda Research Laboratory (Bethesda, Md), and rabbit antitissue polypeptide B 1 (TPA-B 1) from AB Sangtec Medical (Bromma, Sweden). The following mouse monoclonal antibodies have been used: antivimentin, antidesmin, antineurofilament from Eurodiagnostics, and antileukocyte common antigen from DAKO-patis. Peroxidase conjugated rabbit antimouse and swine antirabbit antisera were purchased from DAKO-patts and the ABC staining kits from Vector Laboratories (Burlingame, Calif).
Results Twelve of 22 sarcomas were successfully grafted and transplanted for at least two generations. All xenografted tumors demonstrated a Grade 3 malignancy, and their diagnoses are listed in Table 1. Tumor cell populations of ten of these 12 sarcomas or their transplants had an aneuploid DNA content, in contrast to the unsuccessful ones, which were all, except one, diploid (Table 1). The latency period of the transplants varied from 50 to 349 days. After this period tumor volumes doubled within a period of 1270 days (Table 1). Tumors The most relevant data concerning phenotypic markers and ultrastructure are listed in Tables 2 and
3, respectively. Leiomyosarcoma Two leiomyosarcomas were transplanted. One had a fascicular pattern with pleomorphic areas, the other (no. 2) consisted of (predominantly) uniform elongated cells in a myxoid stroma. Immunoreactivity was found for desmin, laminin, and collagen Type IV. Similar histology and almost identical immunoperox-
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Table 1 -Diagnosis of the Various Human Sarcomas, Relative DNA Content of the Human and Murine Sarcomas, and the Latent Period and Doubling Time of the Murine Sarcomas Case no.
Diagnosis
Successful transplantation 1 Leiomyosarcoma 2 Leiomyosarcoma 3 Malignant schwannoma 4 Malignant schwannoma 5 Synovial sarcoma, biphasic 6 MFH, storiform pleomorphic 7 MFH, idem 8 MFH, idem 9 MFH, idem 10 MFH, idem 11 MFH, myxoid 12 MFH, pleomorphic
Unsuccessful transplantation 13 Mesothelioma 14-22
MFH (2x), fibrosarcoma (1 x), leiomyosarcoma (2x), liposarcoma (3x), angiosarcoma (1 X), agressive fibromatosis (1 x)
Latent periodt (day)
Doubling
1.8 1.0 1.7 1.0
195 210 181 332 203
ND 35 46 ND 48
1.5
112
16
1.6 1.6 2.0 1.0 1.0 (1.6) 1.5
115 295 139 299 349 50
28 31 18 ND 70 12
2.0 1.0
NP NP
NP NP
DNA content* 1.0(1.8)
timet (day)
nd: not done; tumor size could not be measured, or tumors did not yet attain 100 cu mm. NP: not possible; unsuccessful transplantation. * Relative DNA content (DNA index) of the original and xenografted (in parentheses) tumors. Diploid tumors have a DNA index of 1. In Cases 1 and 11 of the original tumors an aneuploid cell population could not be detected, but the DNA content of the xenografted tumors was aneuploid. t Latency period indicating the mean period in which the grafts of the first generation reached a volume of 50 cu mm. $ Mean period of time in which tumor volume of xenografts in the first passage doubled (measured from 50 to 100 cu mm).
idase results were found in the xenografts. As judged by EM, pinocytotic activity of the plasma membrane of the tumor cells was present at various intensities (Table 3). Focal densities of actin filaments were sometimes seen. These two ultrastructural leiomyogenic aspects were more often found in the transplants, especially in tumors of the second and third
generation. Malignant Schwannomas Two cases of neurogenic origin were transplanted (nos. 3 and 4). Both tumors demonstrated a storiform pattern. Pleomorphic elongated cells varied in size. In one tumor (no. 4) a collagenous matrix was present, whereas the other one was more cell dense. The histology of the original and xenografted sarcomas showed a striking resemblance. Immunoperoxidase studies revealed that one tumor lacked immunoreactivity for S100 proteins, but the xenografts derived from it expressed this marker focally. In addition, the cells of these sarcomas were also immunoreactive for lami-
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nin, collagen Type IV, y-enolase and focally for myelin basic protein (Table 2). The presence of long intertwinements of tumor cells was the most remarkable ultrastructural feature. Other features were remnants ofbasal lamina, a moderate amount of dilated rough endoplasmic reticulum, and lysosomes. The tumor cells of the xenograft sarcomas also showed these schwannian features (Table 3).
Synovial Sarcoma One biphasic synovial sarcoma was studied. It showed the classical LM histology, the typical immunoperoxidase pattern, and the characteristic ultrastructure (Tables 2 and 3). Cytokeratin+ epitheloid cells and vimentin+ epitheloid and fibroblastlike cells were detected. Type IV collagen was present around the fibroblastlike cells. Epitheloid cells showed microvillilike cell folds, desmosomelike structures, and tonofilaments. The histologic, ultrastructural, and marker characteristics of the cells in xenografts were similar to those of the cells in the original sarcoma. Table 2-Immunophenotype of the Various Tumors and Their Transplants
Case no.
Original tumor
Leiomyosarcoma 1 Desmin ± (focal),
NSE+, collagen IV + Desmin +, laminin + collagen IV + Malignant schwannoma S-100 ++, laminin + 3 4 NSE + 2
Synovial sarcoma 5 Epithelial-like cells: keratine +, TPA +, fibroblastlike cells: collagen IV + MFH 6 7
8 9
Transplant desmin +, NSE +, collagen IV + desmin +
S-100 + NSE +, S-100 + (focal), laminin +, GFAP +, collagen IV ++, NSE +, MBP + (focal)' Epithelial-like cells: keratine +, TPA + fibroblastlike cells: collagen IV +
NSE + -
desmin + desmin +,t NSE +, collagen IV ±
NSE ± NSE + (focal) -
NSE ± (focal) NSE +
NSE ±, collagen IV+,
GFAP+* 10 11
12
Markers found are listed except for vimentin, which was always present. Markers that were never found are: Factor Vil related antigen, neurofilament, and leukocyte common antigen. Symbols: ±: weak; +: moderate; ++: strong positivity. * One of three xenografts was focally positive for desmin. t Immunoreactivity was found for desmin in the transplants of the second and third passage. t Immunoreactivity was found for NSE and GFAP in the transplants of the second passage but not in those of the first one.
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Table 3-Characteristic EM Features of Non-MFH Sarcomas and Their Transplants Case no.
Tumor
Leiomyosarcoma 1 Original Transplant
2
Original Transplant
Malignant schwannoma 3 Original Transplant 4
Original Transplant
Synovial sarcoma 5 Original
Cell surface Basal lamina ± pinocytotic vesicles + Basal lamina ± pinocytotic vesicles -
Organelles RER +
RER +, short
Inclusions
Other features
Stage of differentiation
Lysosomes + filament condensations ± Lysosomes + Golgi complex + filament condensations ++ lipid - to ± Lysosomes +
Collagen ±
Myogenic (poorly)
Collagen +
Myogenic (moderately)
Pinocytotic vesicles + basal lamina ± Pinocytotic vesicles ± basal lamina ±
RER + RER +
Lysosomes ± filament condensations ++ lipid - to ±
Collagen ±
Myogenic (moderate)
Long external processes + basal lamina ± Long external proces-
RER +
Lysosomes +
Collagen ++
Schwannian (poorly)
RER +
Lysosomes ±
Collagen ++
Schwannian (poorly)
RER ++
Lysosomes + Golgi complex + Lysosomes ± Golgi complex ++
Collagen +
Schwannian (poorly)
Collagen +
Schwannian (moderate)
ses + Long external processes + Long external processes ++ basal lamina ±
RER ++, long
Cell junctions ++ microvilli ++ basal lamina +
RER ±, short
Cell junction ++ microvilli ++ basal lamina +
RER +, short polyribosomes
polyribosomes
Myogenic (poorly)
Tonofilaments ++ glycogen ++
Biphasic: epitheloid and fibroblastlike
Tonofilaments ++ glycogen ++
Biphasic: epitheloid and fibroblastlike cell
++
Transplant
++
MFH neoplasm 6 Original
7
Lysosomes ++
Lysosomes ++ lipid +
-
Fibrohistiobla.stic
Lysosomes ± filament condensations +t
Collagen +
Myogenic
Lysosomes +
-
Fibrohistioblaistic
Lysosomes + filament condensations ±
Contracted nuclei +
Myogenic
Lysosomes ++
Myxoid
Fibrohistioblaistic
RER +
Lysosome - to ++ filament condensation ± to +*
Original
Interdigitating processes +
RER +, Golgi
basal lamina-to ± pinocytotic vesicles - to ± Long extemal processes ± to + basal lamina ± pinocytotic vesicles +++
complex +
Original
-
Fibrohistioblastic Myogenic
Pinocytotic vesicles ++
RER ++, branched Golgi complex ++ RER +, dilated
Col lagen + C ontracted nuclei + ColIllagen +
Transplant
Transplant
8
RER +
Golgi complex +
Transplant
Basal lamina + pinocytotic vesicles +
RER +, dilated
Long extemal proces-
RER +, dilated
Golgi complex ++
9
Original
ses +
Transplant
Long external processes + basal lamina-to ±
Golgi complex ± to + RER ++, short, dilated.
matrix
-
Golgi complex
Original Transplant
External processes +
Long external processes ++ cell contracts-to ± pinocytotic vesicles ±
RER + Golgi
Lysosomes ++
complex + RER ± Golgi complex ++
Lysosomes + glycogen +
secretion vac +
Schwannian
surface
++
10
Amorph deposit along cell
Fibrohistioblaistic
Collagen +
Schwannian
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Table 3-Continued Case no. 11
12
Tumor
Original
Cell surface Microvilli +
Organelles RER ++ (mucoid?)
Transplants
Microvilli + small cell contacts ±
RER ++ dilated (mucoid?) Golgi complex ++
Original
Microvilli +
Transplant
Short microvilli +
RER + Golgi complex + polyribosomes + RER +, Golgi complex + polyribosomes ++
Inclusions
Lysosomes + large vacuoles ++ perinucleair bundles of filaments + Lysosomes + large vacuoles ++ perinuclear bundles of filaments +
Other features
Amorph
Stage of differentiation
Histiocytic and fibroblastic
matrix +
Collagen ±
First generation: histiocytic; second generation: undifferentiated
Lysosomes ++
Collagen +
Histiocytic
Lysosomes +
Collagen ±
Undifferentiated
Symbols: -, featureis absent; ±, feature is sometimes present; +, feature is moderately present; ++, feature is abundantly present. This feature could easly be detected in tumor cells of the transplants of the second and third generation. t These features could easily be detected in tumor cells of the transplants of the third generation. *
Malignant Fibrous Histiocytomas Seven cases of MFH with the following variants were studied: five showed the storiform pleomorphic type, one the myxoid type, and one the pleomorphic type (Table 1). The histologic appearance is illustrated in Figures 1 and 2. As judged from these illustrations, the histologic features of xenografts from the storiform pleomorphic (Cases 6, 7, 9, and 10) and pleomorphic (Case 12) type MFHs were similar to the original ones (Figures 1 A, B, F, and G; Figures 2A and B). In three of these tumors indications for leiomyomatous differentiation were found in the xenografts that were not present in the original sarcoma. In two of them (Cases 6 and 7) immunoreactivity for desmin was seen (Table 2, Figure IC). In all three of these tumors (Cases 6, 7, and 8) ultrastructurally filament condensations and the increased presence of a basal lamina and pinocytotic vesciles were striking findings because they were not found in the original sarcomas (Table 3, Figure 3). Two other cases of MFH showed in the xenografts elements of malignant schwannoma. Long cytoplasmic processes and a focal presence of a basal lamina could be seen around tumor cells in transplants of Case 9. Sarcoma cells of the transplant from Case 10 made some cell contacts and had pinocytotic vesicles and intertwining processes (Table 3, Figure 4). In addition, in Cases 9 and 10 some reactivity for collagen Type IV and GFAP was demonstrated after grafting. Cells of the original sarcoma lacked this type of staining (Figure 2C). Two other cases of MFH did not show signs of differentiation. Cells of the myxoid subtype MFH (Case 1 1) developed broad contact zones with some small plasma membrane contacts. Large dilated
strands of rough endoplasmic reticulum cells were present partly filled with electron dense material (Figure SA). Evidence that those strands are filled with glycosaminoglycans came from the fact that with the LM an intense intracellular staining was seen after incubation with alcian blue. Some lysosomes were present. Cells of the second generation remained classified as undifferentiated polymorphic cells as numerous polyribosomes and filaments were observed (Table 3, Figure 5B). The histiocytelike tumor cells of a pleomorphic MFH (Case 12) were immunoreactive for vimentin only, also after transplantation. As judged by the cytoplasmic organization of cell organelles, an appearance of a primitive cell was revealed (Table 3).
Discussion In agreement with reports on transplantation experiments of STT into nude mice,16-18 we noted a striking similarity of the LM histopathology between the original human tumor and their transplanted counterparts in ten of 12 cases. However, several differences are conspicuous when comparing the ultrastructure and the immunophenotype. In most of the xenografts we found a new or more frequent expression of markers and EM features related to a higher degree of differentiation. For example, the tumor cells of the two human leiomyosarcomas were ultrastructurally characterized as poorly differentiated smooth muscle cells (Table 3). Moderately differentiated smooth muscle cells were seen in the xenografts derived from these leiomyosarcomas; cells of these two cases therefore underwent an increased leiomyogenic differentiation.
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Figure 1-Light microscopy of three storiform pleomorphic MFHs (A, D, F) and their transplants (B, E, G). Case 6 (A-C); Case 8 (D and E); Case 9 (F-H). Large pleomorphic cells and areas with a storiform pattem of short bundles of elongated cells are seen in the human and xenografted tumours of Cases 6 and 9. A pleomorphic sarcoma of large irregularly shaped cells is seen in transplant of Case 8 (second generation), which lacks cellular arrangements in a stonform pattem as is seen in the original tumor (D). Immunoreactivity for desmin is shown in a xenografted tumor of Case 6 (C) (second generation), and for GFAP in a xenografted tumour of Case 9 (H) (first generation). Paraffin sections: A and F, frozen sections: C and H. Burckhard sections: B, D, E, G. All figures: X80, except E: x125.
Xenografts of one of the two cases of malignant schwannomas demonstrated a more mature form and phenotype of schwannian cells, as noted by the EM (Table 3) and also by the expression of markers (Table 2). Thus, more differentiated features came to expression upon xenografting these human types of sarcomas in nude mice. It seemed that three of the five cases with a storiform pleomorphic appearance underwent a leiomyogenic differentiation including immunoreactivity for desmin, whereas the other two demon-
strated a schwannian differentiation. Differentiation marks belonging to lipoblasts were not observed. From these findings and others'6 we conclude that tumor cells of storiform pleomorphic MFHs have the capacity to differentiate into a leiomyogenic, schwannian, or fibroblastic cell lineage. As Brooks23 has already suggested in a hypothetical model of mesenchymal differentiation, it is possible that the cells already were committed to a distinct differentiation lineage and that they differentiated into the various directions
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Figure 2-LM of original and transplant of two different subtypes of MFH. Case 10, a storiform pleomorphic MFH (A-C); Case 11, a myxoid MFH (D-H). Histiocytelike and fibroblastlike cells were present in the original and transplants of Case 10. The cells do not express immunoreactivity for collagen Type IV. An intense staining is present around the vessels (C). Case 1 1 revealed myxoid areas with spindle-shaped cells (D) and some cell dense areas with pleomorphic rounded cells (E). An enlargement of those large mononucleated cells (F). A hemangiopericytomalike pattem without myxoid areas is present in the xenografted tumor of this case (G), with an enlargement in H. Note the resemblance in morphology between the cell dense areas in the original tumor (E and F) and in the xenografted one (G and H). Paraffin section: C; Burckhard sections: other figures. All figures: x80, except F and H x200.
after an appropriate stimulus. Alternatively, it is possible that the growth of the most mature cell forms, already present in the original tumor, was stimulated. However, copious sampling of the original tumor did not provide any indications of these features of differentiation. Another possibility is that very dedifferentiated leiomyosarcomas and malignant schwannomas may regress to the malignant analog of the primitive fibrohistiocytic type of mesenschymal cell. If this supposition is true, we have to assume that after grafting latent possibilities of differentiation may become ap-
parent. This is indeed in line with the features of increases in differentiation that could be demonstrated in leiomyosarcomas and malignant schwannomas after tranplantation. A histogenetic classification of MFH seems almost impossible solely on the basis of LM and EM appearance,24-27 but based on double phenotypic patterns in sarcomas it appeared possible to hypothesize that a fibrohistioblast, which should give rise to MFH neoplasms, is a progenitor cell type common to some other mesenchymal cells.28 The use of cell differentiation markers offers new possibilities to screen stages of the various differentiation lineage.
566
ROHOLL ET AL
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AJP * June 1988
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Figure 3-Elongated cells of a transplant (third generation) derived from Case 6, embedded in a collagen-rich matrix. (x6800) Inset: Pinocytotic vescicles and filamentous condensations, which are ultrastructurally characteristic for a myogenic differentiation. (x25,000)
Figure 4-Cells of a transplant (second generation) derived from Case 10. Ultrastructural characteristics suggesting Schwann cell derivation were observed: long broad, sometimes intertwining cytoplasmic extentions and poorly developed intercellular junctions (arrow head). In addition, lysosomes, some short strands of RER and coated vescicles were seen. (x9200)
The reliability of cytoskeletal proteins as markers for a certain differentiation lineage has been questioned. For example, the expression of GFAP is considered as specific for glial cells, but exceptions have been described for part of the schwannomas.28,29 The immunoreactivity for GFAP of tumor cells with schwannian EM features (Case 9) is therefore not new. The presence of desmin is considered to be restricted to leiomyogenic cells. Positivity was found in two MFH cases for desmin after transplantation, a feature concomitant with the presence of myofibrils. A focal positivity for desmin in one of the three xenografts of the second generation derived from a malignant schwannoma should be considered as an aberrant expression given the ultrastructural characteristics ofschwannian cells. Desmin has been found in some cases of MFH, but it appeared that the number of desmin+ cases of MFH was dependent on type of antibody to desmin used.8 Furthermore, the immunohistochemical detection of neurofilament proteins (70 and 200 KD) in coexpression with vimentin and desmin in part of the
MFH cases has been published.8 We were not able to confirm this observation. NSE is not only present in cells with neuroendocrine differentiation but also in varying amounts in many other cell types.30'3' It seemed that the expression of the y-enolase is increased during malignancy of nonneuroendocrine cells, as was found for astrocytomas.32 Its presence in MFHs, which have a high grade of malignancy, is not surprising, therefore. The ultrastructural presence of a basement membrane or remnants of it is indicative of smooth muscle cells or Schwann's cells. A moderate or weak immunohistochemical expression of collagen Type IV and laminin was reported for leiomyosarcomas and malignant schwannomas, whereas MFHs were found to be negaIn accordance with these reports we found tive. immunoreactivity for these marker proteins in the various types of sarcomas. In addition, immunoreactivity for collagen Type IV was seen in those cases in which a basement membrane was observed ultrastructurally.
567
MALIGNANT FIBROUS HISTIOCYTOMAS
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In conclusion, cells of MFH tumors possess a minimal amount of differentiation characteristics, but under appropriate conditions the storiform pleomorphic subtype appeared to have the capacity to obtain some differentiation features toward a myogenic, schwannian, or fibroblastic stage. Under these conditions the myxoid and pleomorphic subtypes, however, are not capable of developing such signs of differentiation.
References 1. Enzinger FH, Weiss SW: Soft Tissue Tumors. Toronto, C. V. Mosby, 1983, 116-198 2. Du Boulay CEH: Immunohistochemistry of soft tissue tumours: A review. J Pathol 1985, 146:77-94 3. Roholl PJM, De Jong ASH, Ramaekers FCS: Application of markers in the diagnosis of soft tissue tumours. Histopathology 1985, 9:1019-1035 4. Enzinger FM: Malignant fibrous histiocytoma. 20 years after Stout. Am J Surg Pathol 1986, 1O(Suppl):43-53 5. Fletcher CDM: Malignant fibrous histiocytoma? Commentary. Histopathology 1987, 11:433-437 6. Du Boulay CEH: Demonstration of alpha- 1-antitrypsin and alpha- 1-antichymotrypsin in fibrous histiocytomas using the immunoperoxidase technique. Am J Surg Pathol 1982, 6:559-564
7. Brecker ME, Franklin WA: Absence of mononuclear phagocyte antigens in malignant fibrous histiocytoma. Am J Clin Pathol 1986, 86:344-348 8. Lawson CW, Fisher C, Gather KC: An immunohistochemical study of differentiation in malignant fibrous histiocytoma. Histopathology 1987, 11:375-383 9. Roholl PJM, Kleyne J, Elbers H, Van de Vegt H, AlbusLutter CH, Van Unnik JAM: Characterization of tumour cells in malignant fibrous histiocytomas and other soft tissue tumours: I. Immunoperoxidase study on paraffin sections. J Pathol 1985, 147:87-97 10. Roholl PJM, Kleyne J, Van Unnik JAM: Characterization of tumor cells in malignant fibrous histiocytomas and other soft tissue tumors, in comparison with malignant histiocytes: II. Immunoperoxidase study on cryostat sections. Am J Pathol 1985, 121:269-274 11. Strauchen JA, Dimitriu-Bona A: Malignant fibrous histiocytoma. Expression of monocyte/macrophage differentiation antigens detected with monoclonal antibodies. Am J Pathol 1986, 124:303-309 12. Wood GS, Beckstead JH, Turner RR, Hendrickson MP, Kempson RL, Warnke RA: Malignant fibrous histiocytoma tumor cells resemble fibroblasts. Am J Surg Pathol 1986, 10:323-335 13. Iwasaki H, Kikuchi H, Takii M, Enjoji R: Benign and
malignant fibrous histiocytomas of the soft tissue. Functional characterization of the cultured cells. Cancer 1982, 50:520-530
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Acknowledgments The authors would like to acknowledge the expert technical assistance of J. Kleyne, W. T. M. Van Blokland, I. S. Wils, and J. De Frankrijker. We are grateful to Dr. Ch. E. Albus-Lutter (Antoni van Leeuwenhoek Hospital, Amsterdam), Dr. R. S. Menon (Daniel den Hoed Hospital, RRTI, Rotterdam), and Dr. H. A. Van Den Bergen (St. Sophia Hospital, Zwolle), who allowed us to take biopsies of some of the tumors. Furthermore, we wish to thank E. Verbaan and C. Oprel for typing the manuscript.
