Synovial Sarcoma: Anlmmunohistochemical
and Ultrastructural Study
NELSON G, ORDdfiEZ, MD, SOHEIR M. MAHFOUZ, MD, AND 5RlJCE MACKAY, MD Thirty-nine primary synovial sarcomas (15 biphasic, 24 monophasic), amd 19 metastatic synovial sarcomas were studied with a battery of antibodies directed to keratin, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA). vimentin, desmin, muscle-specific actin, smooth muscle actin, S-100 protein, Leu-7, chromogranin A, laminin, collagen IV, Ulex europaeus agglutinin I (UEAI), and the HMB-45 antimelanoma antibody. Twenty-two primary and 18 metastatic synovial sarcomas were also examined by electron microscopy. Epithelial and/or spindle cells in every biphasic tumor, primary and metastatic, reacted for keratin and EMA, but only six primary tumors (five biphasic and one monophasic) showed weak reactivity for CEA which, in the biphasic tumors, was confined to the epithelial component. Of the monophasic tumors, 15 primary (63%) and four metastatic (25%) stained for keratin, whereas seven primary (29%) and two metastatic (13%) tumors reacted for EMA. Only one primary monophasic synovial sarcoma stained for CEA. Tumors thalr stained for EMA or CEA also stained for keratin which is, therefore, the most useful epithelial marker. Immunostaining for epithelial markers, UEAI, collagen IV, and laminin serves to delineate the epithelial component when it is obscure in routine sections. Electron microscopy facilitates the diagnosis when epithelial ,markers are not expressed and aids in separating monophasic synovial sarcomas from other sarcomas that they resemble by light microscopy. HUM PATHOL 21:733-749. 0 1990 by W.B. Saunders Company.
percentage of the pure spindle-cell type, have been reported to stain for keratin and, less frequently, for epithelial membrane antigen (EM A) and carcinoembryonic antigen (CEA). 1.2.4 These observations provide support for evidence from ultrastructural”.’ and tissue culture8 studies relating monophasic synovial sarcoma to the classic biphasic tumor, and indicate that the immunocytochemical detection of these markers could be of great help in establishing the diagnosis of synovial sarcoma when rt is in question. Individually, the three epithelial markers are less than specific. Keratin immunoreactivity is not consistently detected in monophasic spindle-cell synovial sarcomas, and recent reports have demonstrated that it can be found in other mesenchvmal tumors including epithelioid sarcoma,’ ” “).’ ’ leiomvosarconia,‘2.1:~ rhabdomyosarcoma, 14,15 malignant ‘fibrous histiocytoma, IA and some vascular tumors.‘7 Consequently, there is a need to define an effective battery of immunocytochemical markers. It is also pertinent to examine the ultrastructural features of the tumor cells to determine whether they can be relied on to resolve problems in the differential diagnosis. The purpose of this paper is to report our experience with a series of synovial sarcomas studied with a large panel of antibodies, some recently developed, which are known to detect antigens characteristic of epithelial, neuroendocrine, Schwannian, or myogenic cells, as well as some antibodies directed against constituents of the basal lamina. We compare the frequency of expression of these markers in our series of cases with published reports of similar studies in order to better evaluate their diagnostic usefulness. The immunocytochemical findings are correlated with the ultrastructural morphology of the biphasic and monophasic tumors, and we briefly speculate on the histogenesis of synovial sarcoma.
In its classic biphasic form. synovial sarcoma rarely poses a diagnostic problem, but when the epithelioid component of a biphasic synovial sarcoma is difficult to discern or the tumor is a monophasic spindle-cell neoplasm, the diagnosis may be extremely difficult. A monophasic synovial sarcoma can easily be confused with other soft tissue sarcomas which have a similar histologic pattern, including fibrosarcoma, rnalignant schwannoma, hemangiopericytoma, and leiomyosarcoma. Although ultrastructural differences may serve to distinguish between these tumors, it has recently been demonstrated that immunocytochemical markers can be very helpful in the differential diagnosis.‘-” Both the epithelioid and spindle cell components of biphasic synovial sarcomas, as well as a significant
MATERIALS AND METHODS Thirty-nine cases of synovial sarcoma were selected from the surgical pathology files of the M. D. Anderson
From the Department of Pathology, The University of Texas M.D. Anderson Gancer I;enter, Houston. TX. Accepted for publication November 3. 1989. This work was supported by the (;aduceus Foundation. Kc? XW& synovial sarcoma. immunocytochemistry, electron microscopv, kerarin, epithelial membrane antigen, rarcmoembryonic antigen, laminin. collagen IV. S-100 protein, actin, desmin. vimentin. Address correspondence and reprint requests to Nelson G. Ordhfiez, MD, Department of Pathology, Box 85. M. D. Anderson Cancer (:enter. 1515 Holcombe Blvd. Houston, TX 77030. 0 1990 by W.B. Saunders Company. 004fI-x 17719012 107~0009$5.00/0
Center. Cases were included if they fulfilled established histologic criteria,s-go and sufficient paraffin-embedded material for immunocytochemical studies was available. Fifteen of the primary synoviai sarcomas were biphasic and 24 were monophasic. All the primary tumors and metastases were studied with the battery of: antibodies listed in Table 1. The medical records of the patients were reviewed. Cancer
Light Microscopy For light microscopy, tissue sections were stained with hematoxylin and eosin stain and, in selected cases, with pe-
733
HUMANPATHOLOGY TABLE 1. Primary AntibodieLTheir
Volume 21, No. 7 (July 1990) Source, Dilution, and Enzymatic Treatment Animal
Antibody
Source
Vimentin
Dako Corp Santa Barbara, CA Dako Corp Santa Barbara, CA Enzo Biochem New York, NY Dako Corp Santa Barbara, CA Boehringer-Mannheim Indianapolis, IN Boehringer-Mannheim Indianapolis, IN Becton-Dickinson Mountainview, CA Calbiochem San Diego, CA Chemicom El Segundo, CA Instar Stillwater, MN Vector Laboratories Burlingame, CA Dako Corp Santa Barbara, CA Sigma Chemical Co St. Louis, MO Enzo Biochem
Desmin Muscle-specification (HHF35) s-100 Keratin (AE lIAE3) Chromogranin (LK2HlO) Leu-7 Laminin Collagen
IV
CEA UEAI EMA Smooth
muscle
(Type)
actin
HMB-45
Dilution
Enzymatic Digestion
Mouse
(MAb)
:20
No
Mouse
(MAb)
:lOO
No
Mouse
(MAb)
:700
Yes
Rabbit
(PAb)
:I,000
NO
Mouse
(MAb)
:300
Yes
Mouse
(MAb)
:300
No
Mouse
(MAb)
:50
No
Rabbit
(PAb)
1:lOO
Yes
Mouse
(MAb)
I:100
Yes
Rabbit
(PAb)
1:2
NO
Rabbit
(PAb)
1:2,000
Yes
Mouse
(MAb)
1:40
Yes
Mouse
(MAb)
l:l,OOO
No
1:750
Yes
Mouse (MAb)
New York, NY Abbreviations:
MAb. monoclonal
riodic acid-Schiff before blue, and mucicarmine.
antibody;
and after diastase
PAb. polyclonal
digestion,
antibody.
alcian
Immunocytochemistry Antibodies. Antibodies to keratin, EMA, and CEA were used to identify the epithelial phenotype of the tumors. Antibodies to S-100 protein, Leu-7, and chromogranin A were used to investigate possible Schwannian or neuroendocrine differentiation. Antibodies to desmin, musclespecific actin (HHF35), and smooth muscle actin were used to evaluate possible myogenic differentiation. Tissues were stained for collagen IV and laminin to identify basal laminar material associated with the tumor, and with Ulex europaem agglutinin I lectin (UEAI) which is typically bound to endothelial, as well as to some epithelial, cells and tumors derived from these cellszl Specimens were also stained for vimentin, an intermediate cytoskeletal filament which, while considered characteristic for mesenchyma1 differentiation, is also widely distributed in other types of cells and tissues. In addition, sections were stained with the recently developed HMB-45 monoclonal antibody which, in our experience, is specific for melanoma.22 All the antibodies have been well-characterized and can be obtained from commercial sources. Their type, sources, and dilution titers are listed in Table 1. Staining procedure. Immunocytochemical studies were performed on formalin-fixed, paraffin-embedded tissue sections using the avidin-biotin peroxidase complex (ABC) method.23 Tissue specimens were cut 3 to 4 km thick, deparaffinized in xylene, and rehydrated in descending grades (100% to 70%) of ethanol. In order to enhance the immunostaining with some of the antibodies used, the sections were digested with 0.1% protease (type XIV, Sigma
734
Chemical, St Louis, MO) in phosphate buffer at pH 7.6 for 30 minutes. For other sections, this step was omitted since the enzymatic treatment produced a detrimental effect on the immunostaining (Table 1). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in absolute methanol for 10 minutes. Sections were incubated in a humid chamber with primary antibodies for 1 hour at room temperature. This was followed by immunoperoxidase staining using ABC kits (Vector Laboratories, Burlingame, CA). The immunostaining was developed using 3-amino-gethylcarbazole as chromogen. The slides were counterstained with Mayer’s hematoxylin. In order to evaluate the specificity of the antibodies and the effect of enzymatic digestion on the immunostaining, positive and negative control tissue sections were stained as described elsewhere.24
Electron Microscopy The material studied by electron microscopy was comprised of 22 primary tumors and 18 metastases from 34 patients (Table 2). Tissue specimens were fixed in 2% buffered glutaraldehyde, postfixed in 1% osmium tetroxide, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate.
RESULTS Light Microscopy The epithelial component in seven of the biphasic tumors was characterized by columnar or cuboidal cells forming gland-like structures (Fig 1). In one tumor prominent papillae were present. In another the epithelial component lined irregular slit-like spaces as
SYNOVlAL SARCOMA (Ord6riez et al]
TABLE 2. Case No.
~
F/32
2
MINA
3 4
MIN.4 Ml21
5 6 7 x
F/26 F/l.5 Ml 1(I Ml21
9
F/2X
11 I2 13 1-I
I.5 16 Ii
Ml29 Fi27 MI50 F/46 F/33 F/52 F/50
1X
F/17 F/54
19
M/39
20 21 21
Ml28 F/59 X1/2 1
2.? 24 2.i
F/5 1 F/30
26 27 28
Ml29 M/10 F/13
29 30 31
F/3 1 MI48 Ml22
32
M/3X
33
Ml? 1
34
F/20 F/4X Ml?6
37
F/70 Fi54 F/15
‘3, 36
38 39
Location
Sex/Age
1
I0
Location, Type, and lmmunohistochemical
R
leg,
Lung_* L scipular region, Lung* Foot R thigh. Lung* L thigh* L leg* R leg Sacroiliac joint, Lung* L buttock, L lung*, L lung R thifh. Bronchial LN* L leg* R thigh Retroperitoneum* R thigh* Knee Buttock*. Retroperitoneum*t R thigh* R thigh, Lung* L forearm. Lung* L lower leg* R foot* R forearm*, Lung* Pharynx L forearm* L thigh. Lung*, Lung* R leg* .4bdominal wall* Abdominal wall*, Retroperitoneum*# L foot* R post neck* R axilla, Lung* L arm*, Lung* R popliteal area, Lung* L elbow* Neck* R shoulder, Lung*, Lung* R arm* Calf* Sphenoid and palatine fossa*
Type M M M M M B B B M B M M B M M B M B M M M B M M B M M B M M M B B M M M M M B B M M M B M M M M B B M M M M M M B M
KER
EP
3+ 3+ -I+ 4+
4-t
4+ 4+
4+
4+
4+
-I+ 4+
4+ 3+
4+
4+ 4+
4+
EMA ___ SP
2+ 1+ 1+ 0 3+ 3+ 3+ 2+ 0 1+ 1+ 0 0 0 0 1+ 0 4+ 2+ 3+ 2+ 0 1+ 3+ 0 1+ 0 0 4f 0 4+ 1+ 2+ 2+ 0 0 0 0 0 0 1+ 0 3+ 0 1+ 0 1+ 0 0 0 1+ 0 0 0 2+ 3+ 0 2-t
EP
3+ 1+ 4+ 3+
4+
3+ 3+
3+
4+
3+
3+ 1+
4+ 2+
4+
4f 2+
2+
Findings in 39 Cases of Synovial Sarcoma ~
SP 0 0 0 0 1+ 2+ 1+ 1+ 0 1+ 0 0 0 0 0 0 0 1+ 0 0 0 0 0 2+ 0 0 1) 0 ?+ 0 4+ 0 0 3+ 0 0 0 0 3+ 0 0 0 3+ 0 0 0 1+ 0 0 0 1+ 0 0 0 0 2+ 0 0
CEA
EP
~ SP
UEAI
EP
0
0 0 0
0
0
0
0 0 0 0 0
3+ 2t 4t 0
0
0 0
3-t
1-t
0 0 0
2+
0
0
3+
0 0
0
3-t
0
1+ 0
0 0
0
0 0
0
SP
0 0 0 0 1+ 0 1+
~
0 0 0 0 0 0 0 0 0 0 lt 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4+
3+
3+ 3+
4+ 4+
4+
3+ 3+
4t
0 2+ 3+ If 0 0 0 0 0 0 0 0 0 1t 0 0 1+ 0 0 4t 0 0 0 0 3t 0 it 1t 2+ 0 0 0 0 I) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
VIM
EP
It 1t 3t 0
0
0 0
0
-SP 3t 3t 3t -It 2+ 2+ 2+ 4+ 0 0 0 1+ 1+ 1t 3t 3t 4t 0 It 4t 2t It
LE.U-7
0
I+ 2+
0 0
1t
0 0
1t
4t 4t 1+ 0
It 0 I+ It 3+ 4+ 2t 0 It 3t 0 3t 2t 1t 1+ 3t 2t 2t It 0 2t 2t 2t It 4t 2t 3t 3t 2t 2+ 3+
s-100
EP
SP
EP
0 0 0
1t 2+ 0 0 I+ 0 (J 0
0 0 0
0
0
0 0
0
It 0
~
0
0
0 0
0 1
1
0 0
0 0 0
1-t 0 0 0 0 0 0 0 0 0 0 0 0 0 1t 2t 0 0 0 1 0 0 0 0 (I 0 0 1 0 (I 0 0 0 0 0 0 0 0 0
SP
0
0
0 0
0
0
0 0 0 0 0
1+
0 0 0
Notes: 0, no immunostaining; 1 t , 1% to 25% positive cells; 2 t , 26% to 50% positive cells; 3 t , 51 R’ to 75% positive cells: 4 t , >75% positive ~11s. Abbreviations: M, monophasic; B, biphasic; Ep. epithelial; Sp, spindle; KER. keratin; VIM, vimentin; NA, not available, * Ultrastructural studies performed. t Metastasis.
735
HUMAN PATHOLOGY
Volume 21, No. 7 (July 1990)
of gland1Jlar-like and solid patterns in epithelioid areas. The glandular-like FIGURE 1. (Left] Biphasic synovial sarcoma with a combination 1 3n of the same tumor showing keratin immunoreactivity restricted spaces contain proteinaceous material. (Right] lmmunostaining F to the epithelioid component.
lmmunocytochemistry
a single layer of flattened cells (Fig 2, left). In the remaining biphasic tumors, polygonal epithelial cells formed solid clusters. The spindle cell component of these biphasic tumors was made up of slender, fusiform cells with indistinct, tapering cytoplasm. The epithelial component was typically discrete where it formed glandular structures, but in a number of the tumors, particularly those with solid islands of epithelial cells, areas of apparent transition were observed. All the monophasic tumors were made up of densely packed spindle cells that varied in length but appeared shorter than normal fibroblasts. The cells were usually uniform and moderately plump with inconspicuous cytoplasm, and they were compactly grouped (Fig 3, left). There was scantly intercellular collagen in the most cellular areas, but sclerosis with foci of calcification was frequent. The cells were oriented within broad sheets forming bands and, on occasion, interlacing fascicles (Fig 3, right). In one tumor they formed a storiform pattern. In another, whorls of cells contained occasional squamoid areas intermixed with poorly developed glandular-like structures (Fig 4). In four tumors a hemangiopericytic pattern was present (Fig 5). Four monophasic tumors contained numbers of cells with a rhabdoid appearance.
The immunocytochemical results are summarized in Tables 2 and 3. The epithelial component of all 15 biphasic synovial sarcomas stained for keratin, but the spindle cells were positive in only six (Figs I, right and 2, right). Fifteen of the 24 monophasic tumors reacted for keratin (Fig 3, right). Two of the negative cases were recurrences in which previously resected specimens were positive for this marker. The keratin staining of the epithelial component of the tumor was usually diffuse. In contrast, reactivity in the spindle cells of both the biphasic and the monophasic tumors was almost invariably focal. The spindle cells of 20 of the 24 monophasic synovial sarcomas, and of every biphasic tumor, reacted for vimentin. Immunoreactivity for both vimentin and keratin occurred in the epithelial component of five of the 15 biphasic tumors, as well as in the 13 primary monophasic synovial sarcomas which originally stained for keratin. Coexpression of keratin and vimentin occurred not only in different groups of cells but also in the same individual cells. The percentage of cells which coexpressed both markers varied from tumor to tumor (Figs 5, top left and bottom right). In one monophasic tumor which contained 736
SYNOVIAL SARCOMA (Orddtiez et al]
FIGURE 2. [Left) Biphasic synovial sarcoma showing irregular crack Iirled by plump spindle cells. (Right] lmmunostaining preparation demonstrating keratin reactivity of the cells lining the crack-like spaces.
occurred throughout the cytoplasm, it tended to be stronger along the periphery of the spindle cells. All of these cases also reacted for keratin. Only six tumors (five biphasic and one monophasic) reacted for CEA. The number of positive cells staining with this marker was lower than those reacting for keratin or EMA. Fifteen primary monophasic (63%) and four metastatic (25%) spindle-cell tumors reacted for one of the epithelial markers. The marker most commonly detected was keratin, followed by EMA and CEA (Table 3). Thirteen of the 15 biphasic tumors, and two of the 24 monophasic tumors, reacted for UEAI. Staining in the epithelioid areas of these tumors was usually stronger along the periphery of the cells, and was most marked on the luminal surface of the cells lining glandular structures. Material within the lumens of some tumors also reacted for this lectin. Reactivity for Leu-7 occurred in six primary and Iive metastatic spindle-cell monophasic tumors and in the epithelial component of three biphasic sarcomas (Fig 5, bottom right). Immunostaining for S-100 protein occurred in two biphasic tumors and in one monophasic tumor in which reactivity was seen in both the primary tumor
ill-defined whorls of spindle cells, those at the center of the whorl stained strongly for keratin but were negative or weakly positive for vimentin, whereas the peripheral cells reacted strongly for the latter marker. Epithelial areas were rendered easily recognizable by combined keratin and vimentin immunostaining (Figs 4, top right and bottom left). Coexpression of both filaments was also seen in some of the cells located at the periphery of the nodules, indicating that a transition was occurring between the epithelioid and sarcomatoid components (Fig 4, bottom right). All four of the monophasic tumors which presented a. rhabdoid-like appearance reacted strongly for vimentin, but two also coexpressed keratin in a smaller Ipercentage of cells. Immunostaining for EMA was seen in the epithelial component of all biphasic tumors. In the gland-like structures, the staining occurred mainly along the apices of the cells and in the lumens. As a consequence of this staining pattern, lumens that were difficult to identify on routine light microscopy in the sollid tumors in hematoxylin-and-eosin sections could be visualized in the immunoperoxidase preparations. The spindle-cell component of five biphasic tumors reacted for EMA. Seven of the monophasic sarcomas stained for EMA and, although the staining 737
HUMAN PATHOLOGY
Volume 21, No. 7 (July 1990)
FIGURE 3. [Left) Monophasic synovial sarcoma made of short, F)IUIw reacting for keratin.
spindle cells. [Right] Interlacing fascicles of fibroblast-like cells
timelanoma mors.
and the metastasis (case 32). The reaction involved both cytoplasm and nuclei and was usually focal (~30% of the cells); however one tumor tended to stain diffusely (80% of the cells, Fig 6). Although some variability in intensity of staining was seen between laminin and collagen IV, the reaction pattern appeared to be similar. In six biphasic tumors, continuous staining occurred along the epithelial areas. In most of the monophasic tumors, and in the spindle-cell component of the biphasic tumors, focal staining for collagen IV and/or laminin was detected between groups of cells. In the tumors with a hemangiopericytic pattern, unbroken staining occurred along the blood vessels enhancing the distinctive vasculature of the tumor (Fig 5, top left). On occasion, a significant amount of staining was visible around individual tumor cells. No immunoreactivity for any of the muscle markers, or for chromogranin A or the HMB-45 an-
antibody,
was obtained
in any of the tu-
Electron Microscopy In the biphasic tumors, cells of the epithelial component enclosing glandular lumina were connected at the luminal margins by tight junctions of moderate length, and below these junctions by a series of mature desmosomes with short tonoftlaments (Fig 7). Some infolding of lateral cell membranes was common, and filopodia protruded into narrow gaps between lateral cell surfaces. The epithelial cells rested on an intact basal lamina (Fig 8). Microvilli on the apical cell surfaces were often short and irregular in their distribution and length, but in many smaller lumina they were long and curved. Rare cilia were encountered on the epithelial cells and on an occasional spindle cell of three of the biphasic tumors (Fig
FIGURE 4. flop left) Biphasic synovial sarcoma presenting solid nodules of epithelioid areas containing poorly formed duct-like structures. flop right) Solid, well-demarcated epithelioid area reacting for keratin in the same tumor. (Bottom left) lmmunocytochemical preparation showing positive reactivity for vimentin in the sarcomatoid areas while the epithelioid component is negative. [Bottom right) lmmunostaining for vimentin preparation showing a poorly demarcated epithelioid area, suggesting a transition between the epithelioid and the sarcomatoid components of the tumor.
738
739
740
SYNOVIAL SARCOMA (Ord6Aez et al] FIGURE 5. (Top left) Laminin immunostaining accentuating the vascular pattern of a monophasic synovial sarcoma with hemangioperiq-tic pattern. (lop right) lmmunopreparation of the same tumor showing a strong paranuclear reaction for vimentin. (Bottom left) Cluster of cells stairling for keratin. (Bottom right] Cluster of spindle cells reacting for Leu 7.
7) as well as in six monophasic tumors. Some of the lumina contained amorphous material. Occasional small aggregates of glycogen were seen within the cytoplasm of the epithelial cells, but mucin was not found. Mitochondria occasionally filled the apical cytoplasm, lbut usually they were sparse and accompanied by only scattered slender cisternae. The cytoplasm of some cells contained intermediate filaments ranging from a few slender wisps (Fig 9) to diffuse aggregates. Although the filaments were focally condensed, myofilaments were never seen. Epithelial cells forming solid areas lacked tight junctions and microvilli. and had closely apposed cell surfaces with small but sometimes mature desmosomes and at most short tonofilaments. It was common to find small lumina amild apparently solid groups of cells (Fig 10). In the biphasic sarcomas, the spindle cells were usually loosely distributed within a collagenous stroma (Fig 11). but some neighboring cells were joined by small desmosomes or more primitive junctions. The cytoplasm tapered but did not branch. A few cytoplasmic lipid droplets were common, but organelles consisted primarily of a few cisternae that were som’etimes dilated and sparse mitochondria. Ther,e were some similarities between the spindle cells of the biphasic tumors and those of the monophasic sarcomas. Typically, the latter were short and fusiform with nonbranching cytoplasm. The cell surface was usually smooth (a few tumors had cells w-ith frequent filopodia), and intimate contact with neighboring spindle cells over part of a cell surface was the rule (Fig 12). At these sites, small cell attachments, either desmosomes or less structured junctions, were present in small numbers. Where cells were not in contact, slender zones of collagen intervened. These relationships between the cells were evident in longitudinal or oblique sections, and were well-demonstrated when the spindle cells were cut transversely (Fig 13). It was not possible to determine TABLE 3.
lmmunocytochemistry
whether the cells were in separate small groups, or were forming an interconnected meshwork. At some points, cells were separated by a wide band of collagen fibers with intermingled amorphous material similar in consistency and appearance to basal lamina. This material and collagen were abundant in the sclerosed areas of the tumor. A few cells had short, slender cytoplasmic extensions containing some nonspecific intermediate filaments but no myofilaments or arrays of microtubules. One monophasic tumor presented intracellular collagen in spindle tumor cells. Organelles in the monophasic sarcomas were similar m type and number to those within spindle cells in the biphasic tumors. The nuclei were frequently bland in appearance with smooth profiles and fine, evenly distributed chromatin. Nucleoli varied from small to moderate in size, and they were conspicuous against the background of dispersed chromatin. Chromatin clumping did occur in some tumors, and was common when preservation was less than optimal. In the four monophasic tumors containing rhabdoid cells, the globoid inclusions seen on light microscopy were formed by large aggregates of intermediate filaments (Fig 14). DISCUSSION The immunocytochemical findings from this study are in accord with those of previous reports. 1-5.2.5.p(iKeratin is the epithelial marker most often expressed and it can be detected in both the epithelial and spindle cell components. Immunostaining for keratin may, therefore, aid in differentiating a monophasic spindle-cell synovial sarcoma from softtissue neoplasms with similar histopathology. Epithelial membrane antigen and CEA are also expressed in some synovial sarcomas, but in significantlv fewer cases than those which react for keratin: this is parResults
in 39 Synovial
Sarcoma
Cases
Markers
Primary Tumors (n = 39) Biphasic (n = 15) Epithelial component
Keratin @)
EMA 6)
CXA (9%)
15
15 (100)
.i (33) 0
(100) Spindle Monophasic
(Xls (n = 24)
Distant Metastases (n = 19) Biphasic (n = 3) Epithelial component
6
(‘w
(9;)
15 (63)
7 (29)
(4)
3
0
3
( 100) Spindle
cells
2 (67)
Monophasic
(n = 16)
I
UEAl (%)
13
1 (33) 9 (13)
0
4, 12
(“3
(X0)
2 (8)
20 (83)
3
2 (67) 3 (100) 13 (81)
0
741
2 (671 2 (13)
S-100
(8)
1
(87) 4
( 100)
( 100)
Vimentin (s7r)
(7) (1:) 1 (4)
0 0
1 Cf.9
HUMAN PATHOLOGY
Volume 21, No. 7 (July 1990)
FIGURE 6. [Left) Biphasic svnovial sarcoma demonstrating reactivity for S-100 in the epithelioid cells. [Right) Monophasic tumor showing numerous s&dle dells staining for S-100.
in the intensity of the staining correlated with the quantity of intermediate filaments seen by electron microscopy. Since vimentin can also be found in a wide variety of epithelial neoplasms, reactivity for this marker alone has little diagnostic value. However, when coexpressed with other intermediate filaments, it may facilitate the identification of certain neoplasms. An example is the renal rhabdoid tumor,*’ in which the eosinophilic cells contain prominent globular, hyaline inclusions that impart a rhabdomyoblast-like appearance. Ultrastructurally, the inclusions are accumulations of intermediate filaments, and they react for vimentin and keratin but seldom for muscle markers.28~2g So-called malignant rhabdoid tumors have been reported in a variety of extrarenal sites including soft tissues, but Tsuneyoshi et a130 were able to identify rhabdoid cells in a variety of specific soft-tissue sarcomas including epithelioid sarcomas, extraskeletal myxoid chondrosarcomas, and synovial sarcomas. It seems likely that extrarenal malignant rhabdoid tumors are a heterogeneous group with only a morphologic resemblance to the renal neoplasm. All four of the tumors with rhabdoid features in our series coexpressed vimentin and keratin, and the staining was concentrated in the cytoplasmic inclusions. Ultrastructurally, the intermediate filaments in
titularly the case with the monophasic tumors. In our study, EMA immunostaining occurred in seven of 24 primary monophasic tumors and in two of 16 metastatic monophasic tumors. Although all of our cases that stained for EMA also reacted for keratin, EMApositive, keratin-negative monophasic tumors have been reported.’ Therefore, EMA staining should be performed on suspected synovial sarcomas when keratin staining is not contributory. Only six of the synovial sarcomas in our series reacted for CEA, and the staining was focal and confined, in five of the cases, to the epithelial elements of the biphasic tumors, a confirmation of earlier observations.‘.* Corson et al* found reactivity for CEA in three of eight biphasic tumors, localized in the epithelial component, and in only one of 16 monophasic tumors. As was the case in our series, all the tumors in their study that stained for CEA also reacted for keratin. Based on these observations, we believe that CEA has limited value in the diagnosis of synovial sarcoma. Since the tumors in our series that reacted for EMA and CEA also stained for keratin, the last appears to be the epithelial marker of choice. Reactivity for vimentin was obtained in the spindle cells of all the tumors in our series, both biphasic and monophasic. The epithelial cells also stained for vimentin in five of the 15 biphasic tumors. Variations 742
SYNOVIAL SARCOMA (Ord6riez et al)
FIGURE 7. A small lumen from a biphasic synovial sarcoma contains irregular. short microvilli and occasional cilia. The tight junctions and desmosomes are evident. (Magnification x 21,000.)
Recent studies have indicated that antibodies to Leu-7 can recognize an antigenic constituent of myelin-associated glycoprotein and they may. therefore, have some potential in separating neurogenic sarcoma from other spindle-cell sarcomas, including monophasic synovial sarcoma.““.q’l That we were able to identify reactivity for Leu-7 in 11 synovial sarcomas confirms that this marker cannot reliably separate a monophasic synovial sarcoma from a Schwann cell tumor.fi S- 100 protein is known to be expressed in a number of malignant schwannomas,4i-“” while synovial sarcomas have been reported to be negative for this marker.1”4 It has been suggested that staining for S- 100 could be used to distinguish monophasic and biphasic synovial sarcomas from malignant Schwann cell tumors, including those with glandular differentiation.1,“4 This argument cannot be sustained in the light of our observation of S-100 immunoreactivity in both the epithelial and the spindle-cell elements of some synovial sarcomas. Collagen IV and laminin are two major components of the basal lamina that can be demonstrated by immunocytochemical methods, and it has been reported that the immunostaining pattern for laminin can help in the diagnosis of certain soft-tissue tumors.45 We were able to demonstrate continuous reactivity for collagen IV and laminin along the epithelial component of biphasic tumors, and the immunostaining of these substances was helpful in revealing and defining the extent of epithelial areas that were
the inclusions did not display specific features, although they occasionally condensed into bundles resembling the cytokeratin of squamous cell carcinomas. Inclusions composed of intermediate filaments have been reported in a variety of epithelial”‘-33 and mesenchylmal’ ‘zN~’ tumors, as well as in melanomas,‘15 meningiomas,:‘” and mesotheliomas.:” Even nonspecific filaments may contribute to the diagnosis of a tumor in which they occur frequently, notably neuroendoepithelioicl sarcomas’ l and cutaneous crine (Merkel cell) carcinomas.31,““.3s The inclusions may also serve to distinguish monophasic synovial sarcoma from hemangiopericytoma. It has been our experience that the latter does not express keratin or possess large accumulations of intermediate filaments. Synovial sarcoma can potentially be confused with a nulmber of other defined sarcomas, particularly with leiomyosarcoma, neurogenic sarcoma, fibrosarcoma, and malignant fibrous histiocytoma. While a positive reaction for keratin may offer support for a diagnosis of synovial sarcoma, its contribution is diminished when leiomyosarcoma is included in the differential diagnosis, since some leiomyosarcomas have been reported to stain for keratin and EMA.12 Therefore, immunostaining for muscle markers such as smooth-muscle actin or desmin 12,13 The two antibodies to should also be performed. actin used in our project react strongly in paraffinembedded tissue and are more sensitive than desmin in the diagnosis of leiomyosarcomas. 743
HUh4AN PATHOLOGY
Volume 21, No. 7 (July 1990)
FIGURE 8. The epifhelial rest on an intact
Cells
X 7,000.)
x 9,500.)
SYNOVIAL SARCOMA (Ord6riez et al]
FIGURE IO. Narrow lumina within a solid area of epithelial cells. (Magnification x 4,500.)
obscure in hematoxvlin-and-eosin preparations. Immunostaining for collagen IV and laminin in the spindle cells of the biphasic and the monophasic tumors was limited to small foci, and usually correlated with the observation of scanty basal lamina by electron microscopy. The reaction contrasted markedly with the diffuse staining for laminin surrounding individual ‘cells that has been reported in leiomyosarcomas and in some Schwann cell tumors.q” In our study, the demonstration of keratin immunoreactivity often facilitated the diagnosis of monophasic synovial sarcoma, but there remained a significant number of tumors (37% of the primary and 75% of the metastatic monophasic synovial sarc-omas) t!hat failed to express epithelial markers or, with the exception of vimentin, any of the other markers used in the study. Various reasons can be suggested. The marker may have been lost, as probably occurred in six cases of monophasic spindle-cell tumors in which none could be demonstrated in the recurreme or metastasis (Table 2). It is also possible that variation in the sample could account for the staining being focal and confined to a very small number of cells. In thle differential diagnosis of monophasic synovial sarcoma, separation from a primitive, unclassified sarcoma may not be possible, but defined sarcomas with comparable histopathology can usually be identified from their combined light- and electronmicroscopic features.
Compared with the cells of a fibrosarcoma, the spindle cells of a monophasic synovial sarcoma are shorter, and their cytoplasm tapers more sharply and does not branch. Neoplastic fibroblasts do not generally show such consistent close contact of neighboring cell membranes within small clusters of intimately apposed cells, nor do they possess basal lamina. Where collagen intervenes between cells in a monophasic synovial sarcoma, it frequently forms delicate partitions. The cytoplasm tends to have fewer organelles, especially rough endoplasmic reticulum, than ftbrosarcoma cells, and myofibroblastic differentiation is unique to true fibroblastic tumors. ‘The nuclei in monophasic synovial sarcomas are characteristically oval with smooth profiles and fine, evenly dispersed chromatin. Malignant fibrous histiocytoma cells have organelles similar to those in fibrosarcoma, although the cytoplastn is often more extensive and it may contain diffuse zones of intermediate filaments. Intracytoplasmic collagen, which was present in one of our cases, has been reported in various types of sarcomas, including malignant fibrous histiocytomas-l” There are some similarities between monophasic synovial sarcoma and stromal sarcomas of uterus and breast, but the clinical findings usually prevent confusion. Nerve sheath sarcomas display a broad spectrum of histopathology, but ultrastructurally the cells often have long cytoplasmic extensions of uniform caliber that contain filaments and microtubules, and mesaxon formation may be found.+7,AS The spindle745
HUMAN PATHOLOGY
Volume 21. No. 7 (JulY 1990)
FIGURE 11. Biphasic synovial sarcoma in which there is o clear demarcation between the epithelial cells forming the glandular structure and the adjacent spindle cells, many of which contain lipid droplets, (Magnification x 2,800.)
FIGURE 12. The plane of section passes obliquely through spindle ceils of a monophasic synovial sarcoma. Some cells are in direct contact with their neighbors, while others are separated by slender bands of collagen, (Magnification x 4,200.)
746
SYNOVIAL SARCOMA (Ord6riez et al]
FIGURE 13. Cells of the same tumor seen in Figure 6 sectioned transversely. Intimate apposition of cells within tiny bundles is highlighted by the delicate collagen partitions. (Magnification )i 4,200.)
cell component of the rare malignant schwannoma with epithelial differentiation has schwannian features. Malignant melanoma forming in soft tissues, formerly termed clear-cell sarcoma, can resemble a monophasic synovial sarcoma, but the cells are often rounder, nucleoli are typically large and dense, premelanosomes are present, and spindle-cell tumors may manifest some neural differentiation.-‘!’ The histogenesis of synovial sarcoma remains an intriguing question. For many years, the tumors were believed to originate from synovial cells, but when ultrastructural studies failed to show obvious synovial features in the tumors,5”.“’ doubt was cast on this theory. The immunocytochemical evidence does not indicate a histogenetic relationship between the tumors and synovial lining cells. Synovial cells do not express epithelial markers or bind UEAI lectin, whereas synovial sarcomas may do both.‘.*” Since synovial sarcomas express epithelial markers such as keratin, EMA, and CEA, and possess well-developed desmosomes which have been reported to react to desmosomal proteins exclusively associated with epithelial cell~.~~ some investigators have proposed that synovial sarcoma is a unique form of carcinosarcoma confined1 to soft tissues.25.5” Genuine carcinosarcomas are uncommon tumors, often difficult to diagnose with certainty, although some may show specific differentiation m the sarcomatous component, as in the leiomyosarcomatous portion of renal-cell carcinosarcomas.“” It is becoming increasingly recognized that there is not always a rigid separation of epithelial from mes-
enchymal cells. Morphologic distinctions between the two are often blurred. We have. for example, been impressed with the ultrastructural resemblance of sarcomatoid squamous carcinoma cells to fibroblasts. The myoepithelial cell is a hybrid with epithelial and smooth-muscle features which can be variably expressed in neoplasms. Transitions from an epithelial to a mesenchymal phenotype are well-documented in vertebrate development, and are observed in in vitro models.“” Gene expression can be modified by environmental conditions and lead to profound alterations in cell structure: mouse sarcoma cells transfected with complementary DNA encoding chick LCAM assume an epithelial morphology.“” A genetic abnormality in synovial sarcoma has been reported on a number of occasions.5T In spite of light-microscopic similarities between biphasic synovial sarcomas and the synovial membrane, it has not been established that the tumors are displaying authentic synovial differentiation.“*38 The disparate anatomic locations of the primary tumors, often far removed from the nearest articular surface, and the infrequency with which a joint surface is clearly involved, are evidence against sarcomas being truly synovial. The synovial lining is composed of thinly stratified and looselv grouped cells with slender cytoplasmic extensions that arborize over the surface, and desmosomes are notably absent.:” When the tumor cells are compared with those of the normal synovial lining (tendon sheath appears to have a similar construction), there are no obvious resemblances between the two. An analogy with mesothelioma can be seen. Ex747
HUMAN PATHOLOGY
Volume 21, No. 7 (July 1990)
FIGURE 14 A diffuse aggregate of intermediate filaments displaces organelles to the peripheral cytoplasm giving the cell a rhabdoid appearance on light microscopy. (Magnification x 23,000.)
perimental studies have shown that the subserosal spindle cell responds to injury of the overlying epithelium by proliferating, enlarging, assuming first low-molecular weight then high-molecular weight keratin immunoreactivity, and repairing breaches in the mesothelial surface.5Y.6” These observations have led to the postulate that the spectrum of histopathology displayed by mesotheliomas can be accounted for by varied differentiation of the submesothelial spindle cell towards fibroblasts, epithelial (mesothelial) cells, or intermediate forms that lack the extensive endoplasmic reticulum of the mature fibroblasts or myofilaments of a myofibroblast, and also do not possess the basal lamina, complex cell attachments, or luxurious microvilli of the surface cells. The occurrence of the uncommon mixed variant of mesothelioma, in which spaces lined by a single layer of epithelium are interspersed throughout a background of spindle cells, can be explained by this hypothesis. In like manner, a synovial sarcoma could be postulated to develop from a primitive mesenchymal with the inherent or acquired ability to form surface epithelium.
a combined peroxidase-antiperoxidase/avidin-biotin-peroxidase complex procedure. HUM PATHOL 17:1107-l 115, 1986 2. Corson JM, Weiss LM, Banks-Schlegel SP. et al: Keratin proteins and carcinoembryonic antigen in synovial sarcomas: An immunohistochemical study of 24 cases. HUM PATHOL 15:615-621, 1984 3. Carson JM, Weiss LM, Banks-Schlegel SP, et al: Keratin proteins in synovial sarcoma. Am J Surg Pathol 7:107-109, 1983 4. Fisher C: Synovial sarcoma: Ultrastructural and immunohistochemical features of epithelial differentiation in monophasic and biphasic tumors. HUM PATHOL 17:996-1008, 1986 5. Miettinen M, Veli-Pekka L, Virtanen I: Monophasic synovial sarcoma of spindle-cell type. Epithelial differentiation as revealed by ultrastructural features, content of prekeratin and binding of peanut agglutinin. Virchows Arch [B] 44: 187-199, 1983 6. Swanson PE. Manivel IC. Wick MR: Immunoreactivitv for Leu-7 in neurofibrosarcoma and’other spindle cell sarcomas of soft tissue. Am J Pathol 126:546-560, 1987 7. Krall RA, Kostianovsky M, Patchefsky AS: Synovial sarcoma. A clinical, pathological, and ultrastructural study of 26 cases supporting the recognition of a monophasic variant. Am J Surg Pathol 5:137-151, 1981 8. Alvarez-Fernandez E, Escalona-Zapata J: Monophasic mesenchymal synovial sarcoma: Its identification by tissue culture. Cancer 47:628-635, 1981 9. Daimaru Y, Hashimoto H, Tsuneyoshi M, et al: Epithelial profile of epithelioid sarcoma. An immunohistochemical analysis of eight cases. Cancer 59:134-141, 1987 10. Manivel JC, Wick MR, Dehner LP, et al: Epithelioid sarcoma. An immunohistochemical study. Am J Clin Pathol 87:319326, 1987 11. Meis JM, Mackay B, Ord6tiez NC: Epithelioid sarcoma: An immunohistochemical and ultrastructural study. Surg Pathol 1:13-31, 1988 12. Miettinen M: Immunoreactivity for cytokeratin and epithelial membrane antigen in leiomyosarcoma. Arch Pathol Lab Med 112:637-640, 1988
Acknowledgment. The authors wish to thank Thomas E. Brooks, HT, Hazel Dalton, HT, Sharon Thompson, HT. and Elsa Ramos, HT, for their technical assistance.
REFERENCES coma:
1. Abenoza P, Manivel Ultrastructural study
JC. Swanson PE, et al: Synovial sarand immunohistochemical analysis by
748
SYNOVIAL SARCOMA (Ord6riez
13. Norton AJ, Thomas JA, Isaacson PG: Cytokerdtin-specific monoclonal antibodies are reactive with tumours of smooth muscle derivation. An immunocytochemical and biochemical study using antibodies to intermediate filament cytoskeletal proteins. Histopathology 11:487-4!)9, 1987 14. Coidre J-M, De Mascarel A. Trojan MM,et al: Immunohistochemical study of rhabdomyosarcoma. Unexpected staining with S-100 protein and cytokeratin. J Pathol 155: 127-132. I988 1.5. Miettinen M, Rapola J: Immunohistochemical spectrum of rhabdomvosarcoma and rhabdomyosarcoma-like tumors. Expression of cytokeratin and 6X-KD neurofilament keratins. Am J Surg I’dthol 13:120-132. 1989 16. WlGss SW, Bratthauer GL, Morris PA: Postradiation mahgnant fibrous histiocytoma expressing cytokeratin. Implications lor the immunodiagnosis of sarcoma. Am J Surg Pathol 12:554.55X. I988 17. Grav M. Rosenberg .4, Bhan A. et al: Cytokeratin expression bv ephthelioid vascular neoplasms. Lab Invest 60:34A. 1989 (abstr) IX. Enzinger FM. Weiss SW: Synovial sarcoma, in Soft Tissue ‘l‘umors. St Louis, MO. Mosby. 1988, pp 659-688 19. Evans H: Svnovial sarcoma. A study of 23 biphasic and 17 probably monophasic examples. Pathol Annu 2:309-33 1, 1980 20. Hajdu SI. Shiu MH. Fortner JG: Tendosynovial sarcoma. .\ clinicopathological stud, of 136 cases. Cancer 39: 1201-1217. 1977 2 I Ord6Aez NC;.Batsakis JG: Comparisons of c’lrx ~uropaeu\ I lectin and factor VIII-related antigen in vascular lesions. Arch Pathol Lab Med 10X:129-132. 1984 22. Ordbiier IiG. Ziaolong J, Hickey R: Comparison of HMB45 monoclonal antibody and S-100 protein in the immunohistochemical diagnosis of melanoma. Am .J Clin Pathol 90:385-390, 19X8 23. Hsu S-M. Raine L, Fanger H: Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580, 1981 24. Ord6iiez NC;. Manning J, Brooks T: Effect of trypsinizarion on the immunostaining of formalin-fixed. paraffin-embedded rissues. Am J Surg Pathol 12:121-329. I988 25, Miettinen M, \ lrtanen I: Synovial sarcoma--:\ misnomer. * .\m J Pathol I1 /: IX- 2 5”3 1984 26. Salisbury JR, Isaacson PC;: Synovial sarcoma: An immunohistochemical study. J Pathol 147:49-57. 1985 27. Sotelo-Avila
and Ultrastructural Study
NELSON G, ORDdfiEZ, MD, SOHEIR M. MAHFOUZ, MD, AND 5RlJCE MACKAY, MD Thirty-nine primary synovial sarcomas (15 biphasic, 24 monophasic), amd 19 metastatic synovial sarcomas were studied with a battery of antibodies directed to keratin, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA). vimentin, desmin, muscle-specific actin, smooth muscle actin, S-100 protein, Leu-7, chromogranin A, laminin, collagen IV, Ulex europaeus agglutinin I (UEAI), and the HMB-45 antimelanoma antibody. Twenty-two primary and 18 metastatic synovial sarcomas were also examined by electron microscopy. Epithelial and/or spindle cells in every biphasic tumor, primary and metastatic, reacted for keratin and EMA, but only six primary tumors (five biphasic and one monophasic) showed weak reactivity for CEA which, in the biphasic tumors, was confined to the epithelial component. Of the monophasic tumors, 15 primary (63%) and four metastatic (25%) stained for keratin, whereas seven primary (29%) and two metastatic (13%) tumors reacted for EMA. Only one primary monophasic synovial sarcoma stained for CEA. Tumors thalr stained for EMA or CEA also stained for keratin which is, therefore, the most useful epithelial marker. Immunostaining for epithelial markers, UEAI, collagen IV, and laminin serves to delineate the epithelial component when it is obscure in routine sections. Electron microscopy facilitates the diagnosis when epithelial ,markers are not expressed and aids in separating monophasic synovial sarcomas from other sarcomas that they resemble by light microscopy. HUM PATHOL 21:733-749. 0 1990 by W.B. Saunders Company.
percentage of the pure spindle-cell type, have been reported to stain for keratin and, less frequently, for epithelial membrane antigen (EM A) and carcinoembryonic antigen (CEA). 1.2.4 These observations provide support for evidence from ultrastructural”.’ and tissue culture8 studies relating monophasic synovial sarcoma to the classic biphasic tumor, and indicate that the immunocytochemical detection of these markers could be of great help in establishing the diagnosis of synovial sarcoma when rt is in question. Individually, the three epithelial markers are less than specific. Keratin immunoreactivity is not consistently detected in monophasic spindle-cell synovial sarcomas, and recent reports have demonstrated that it can be found in other mesenchvmal tumors including epithelioid sarcoma,’ ” “).’ ’ leiomvosarconia,‘2.1:~ rhabdomyosarcoma, 14,15 malignant ‘fibrous histiocytoma, IA and some vascular tumors.‘7 Consequently, there is a need to define an effective battery of immunocytochemical markers. It is also pertinent to examine the ultrastructural features of the tumor cells to determine whether they can be relied on to resolve problems in the differential diagnosis. The purpose of this paper is to report our experience with a series of synovial sarcomas studied with a large panel of antibodies, some recently developed, which are known to detect antigens characteristic of epithelial, neuroendocrine, Schwannian, or myogenic cells, as well as some antibodies directed against constituents of the basal lamina. We compare the frequency of expression of these markers in our series of cases with published reports of similar studies in order to better evaluate their diagnostic usefulness. The immunocytochemical findings are correlated with the ultrastructural morphology of the biphasic and monophasic tumors, and we briefly speculate on the histogenesis of synovial sarcoma.
In its classic biphasic form. synovial sarcoma rarely poses a diagnostic problem, but when the epithelioid component of a biphasic synovial sarcoma is difficult to discern or the tumor is a monophasic spindle-cell neoplasm, the diagnosis may be extremely difficult. A monophasic synovial sarcoma can easily be confused with other soft tissue sarcomas which have a similar histologic pattern, including fibrosarcoma, rnalignant schwannoma, hemangiopericytoma, and leiomyosarcoma. Although ultrastructural differences may serve to distinguish between these tumors, it has recently been demonstrated that immunocytochemical markers can be very helpful in the differential diagnosis.‘-” Both the epithelioid and spindle cell components of biphasic synovial sarcomas, as well as a significant
MATERIALS AND METHODS Thirty-nine cases of synovial sarcoma were selected from the surgical pathology files of the M. D. Anderson
From the Department of Pathology, The University of Texas M.D. Anderson Gancer I;enter, Houston. TX. Accepted for publication November 3. 1989. This work was supported by the (;aduceus Foundation. Kc? XW& synovial sarcoma. immunocytochemistry, electron microscopv, kerarin, epithelial membrane antigen, rarcmoembryonic antigen, laminin. collagen IV. S-100 protein, actin, desmin. vimentin. Address correspondence and reprint requests to Nelson G. Ordhfiez, MD, Department of Pathology, Box 85. M. D. Anderson Cancer (:enter. 1515 Holcombe Blvd. Houston, TX 77030. 0 1990 by W.B. Saunders Company. 004fI-x 17719012 107~0009$5.00/0
Center. Cases were included if they fulfilled established histologic criteria,s-go and sufficient paraffin-embedded material for immunocytochemical studies was available. Fifteen of the primary synoviai sarcomas were biphasic and 24 were monophasic. All the primary tumors and metastases were studied with the battery of: antibodies listed in Table 1. The medical records of the patients were reviewed. Cancer
Light Microscopy For light microscopy, tissue sections were stained with hematoxylin and eosin stain and, in selected cases, with pe-
733
HUMANPATHOLOGY TABLE 1. Primary AntibodieLTheir
Volume 21, No. 7 (July 1990) Source, Dilution, and Enzymatic Treatment Animal
Antibody
Source
Vimentin
Dako Corp Santa Barbara, CA Dako Corp Santa Barbara, CA Enzo Biochem New York, NY Dako Corp Santa Barbara, CA Boehringer-Mannheim Indianapolis, IN Boehringer-Mannheim Indianapolis, IN Becton-Dickinson Mountainview, CA Calbiochem San Diego, CA Chemicom El Segundo, CA Instar Stillwater, MN Vector Laboratories Burlingame, CA Dako Corp Santa Barbara, CA Sigma Chemical Co St. Louis, MO Enzo Biochem
Desmin Muscle-specification (HHF35) s-100 Keratin (AE lIAE3) Chromogranin (LK2HlO) Leu-7 Laminin Collagen
IV
CEA UEAI EMA Smooth
muscle
(Type)
actin
HMB-45
Dilution
Enzymatic Digestion
Mouse
(MAb)
:20
No
Mouse
(MAb)
:lOO
No
Mouse
(MAb)
:700
Yes
Rabbit
(PAb)
:I,000
NO
Mouse
(MAb)
:300
Yes
Mouse
(MAb)
:300
No
Mouse
(MAb)
:50
No
Rabbit
(PAb)
1:lOO
Yes
Mouse
(MAb)
I:100
Yes
Rabbit
(PAb)
1:2
NO
Rabbit
(PAb)
1:2,000
Yes
Mouse
(MAb)
1:40
Yes
Mouse
(MAb)
l:l,OOO
No
1:750
Yes
Mouse (MAb)
New York, NY Abbreviations:
MAb. monoclonal
riodic acid-Schiff before blue, and mucicarmine.
antibody;
and after diastase
PAb. polyclonal
digestion,
antibody.
alcian
Immunocytochemistry Antibodies. Antibodies to keratin, EMA, and CEA were used to identify the epithelial phenotype of the tumors. Antibodies to S-100 protein, Leu-7, and chromogranin A were used to investigate possible Schwannian or neuroendocrine differentiation. Antibodies to desmin, musclespecific actin (HHF35), and smooth muscle actin were used to evaluate possible myogenic differentiation. Tissues were stained for collagen IV and laminin to identify basal laminar material associated with the tumor, and with Ulex europaem agglutinin I lectin (UEAI) which is typically bound to endothelial, as well as to some epithelial, cells and tumors derived from these cellszl Specimens were also stained for vimentin, an intermediate cytoskeletal filament which, while considered characteristic for mesenchyma1 differentiation, is also widely distributed in other types of cells and tissues. In addition, sections were stained with the recently developed HMB-45 monoclonal antibody which, in our experience, is specific for melanoma.22 All the antibodies have been well-characterized and can be obtained from commercial sources. Their type, sources, and dilution titers are listed in Table 1. Staining procedure. Immunocytochemical studies were performed on formalin-fixed, paraffin-embedded tissue sections using the avidin-biotin peroxidase complex (ABC) method.23 Tissue specimens were cut 3 to 4 km thick, deparaffinized in xylene, and rehydrated in descending grades (100% to 70%) of ethanol. In order to enhance the immunostaining with some of the antibodies used, the sections were digested with 0.1% protease (type XIV, Sigma
734
Chemical, St Louis, MO) in phosphate buffer at pH 7.6 for 30 minutes. For other sections, this step was omitted since the enzymatic treatment produced a detrimental effect on the immunostaining (Table 1). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in absolute methanol for 10 minutes. Sections were incubated in a humid chamber with primary antibodies for 1 hour at room temperature. This was followed by immunoperoxidase staining using ABC kits (Vector Laboratories, Burlingame, CA). The immunostaining was developed using 3-amino-gethylcarbazole as chromogen. The slides were counterstained with Mayer’s hematoxylin. In order to evaluate the specificity of the antibodies and the effect of enzymatic digestion on the immunostaining, positive and negative control tissue sections were stained as described elsewhere.24
Electron Microscopy The material studied by electron microscopy was comprised of 22 primary tumors and 18 metastases from 34 patients (Table 2). Tissue specimens were fixed in 2% buffered glutaraldehyde, postfixed in 1% osmium tetroxide, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate.
RESULTS Light Microscopy The epithelial component in seven of the biphasic tumors was characterized by columnar or cuboidal cells forming gland-like structures (Fig 1). In one tumor prominent papillae were present. In another the epithelial component lined irregular slit-like spaces as
SYNOVlAL SARCOMA (Ord6riez et al]
TABLE 2. Case No.
~
F/32
2
MINA
3 4
MIN.4 Ml21
5 6 7 x
F/26 F/l.5 Ml 1(I Ml21
9
F/2X
11 I2 13 1-I
I.5 16 Ii
Ml29 Fi27 MI50 F/46 F/33 F/52 F/50
1X
F/17 F/54
19
M/39
20 21 21
Ml28 F/59 X1/2 1
2.? 24 2.i
F/5 1 F/30
26 27 28
Ml29 M/10 F/13
29 30 31
F/3 1 MI48 Ml22
32
M/3X
33
Ml? 1
34
F/20 F/4X Ml?6
37
F/70 Fi54 F/15
‘3, 36
38 39
Location
Sex/Age
1
I0
Location, Type, and lmmunohistochemical
R
leg,
Lung_* L scipular region, Lung* Foot R thigh. Lung* L thigh* L leg* R leg Sacroiliac joint, Lung* L buttock, L lung*, L lung R thifh. Bronchial LN* L leg* R thigh Retroperitoneum* R thigh* Knee Buttock*. Retroperitoneum*t R thigh* R thigh, Lung* L forearm. Lung* L lower leg* R foot* R forearm*, Lung* Pharynx L forearm* L thigh. Lung*, Lung* R leg* .4bdominal wall* Abdominal wall*, Retroperitoneum*# L foot* R post neck* R axilla, Lung* L arm*, Lung* R popliteal area, Lung* L elbow* Neck* R shoulder, Lung*, Lung* R arm* Calf* Sphenoid and palatine fossa*
Type M M M M M B B B M B M M B M M B M B M M M B M M B M M B M M M B B M M M M M B B M M M B M M M M B B M M M M M M B M
KER
EP
3+ 3+ -I+ 4+
4-t
4+ 4+
4+
4+
4+
-I+ 4+
4+ 3+
4+
4+ 4+
4+
EMA ___ SP
2+ 1+ 1+ 0 3+ 3+ 3+ 2+ 0 1+ 1+ 0 0 0 0 1+ 0 4+ 2+ 3+ 2+ 0 1+ 3+ 0 1+ 0 0 4f 0 4+ 1+ 2+ 2+ 0 0 0 0 0 0 1+ 0 3+ 0 1+ 0 1+ 0 0 0 1+ 0 0 0 2+ 3+ 0 2-t
EP
3+ 1+ 4+ 3+
4+
3+ 3+
3+
4+
3+
3+ 1+
4+ 2+
4+
4f 2+
2+
Findings in 39 Cases of Synovial Sarcoma ~
SP 0 0 0 0 1+ 2+ 1+ 1+ 0 1+ 0 0 0 0 0 0 0 1+ 0 0 0 0 0 2+ 0 0 1) 0 ?+ 0 4+ 0 0 3+ 0 0 0 0 3+ 0 0 0 3+ 0 0 0 1+ 0 0 0 1+ 0 0 0 0 2+ 0 0
CEA
EP
~ SP
UEAI
EP
0
0 0 0
0
0
0
0 0 0 0 0
3+ 2t 4t 0
0
0 0
3-t
1-t
0 0 0
2+
0
0
3+
0 0
0
3-t
0
1+ 0
0 0
0
0 0
0
SP
0 0 0 0 1+ 0 1+
~
0 0 0 0 0 0 0 0 0 0 lt 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4+
3+
3+ 3+
4+ 4+
4+
3+ 3+
4t
0 2+ 3+ If 0 0 0 0 0 0 0 0 0 1t 0 0 1+ 0 0 4t 0 0 0 0 3t 0 it 1t 2+ 0 0 0 0 I) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
VIM
EP
It 1t 3t 0
0
0 0
0
-SP 3t 3t 3t -It 2+ 2+ 2+ 4+ 0 0 0 1+ 1+ 1t 3t 3t 4t 0 It 4t 2t It
LE.U-7
0
I+ 2+
0 0
1t
0 0
1t
4t 4t 1+ 0
It 0 I+ It 3+ 4+ 2t 0 It 3t 0 3t 2t 1t 1+ 3t 2t 2t It 0 2t 2t 2t It 4t 2t 3t 3t 2t 2+ 3+
s-100
EP
SP
EP
0 0 0
1t 2+ 0 0 I+ 0 (J 0
0 0 0
0
0
0 0
0
It 0
~
0
0
0 0
0 1
1
0 0
0 0 0
1-t 0 0 0 0 0 0 0 0 0 0 0 0 0 1t 2t 0 0 0 1 0 0 0 0 (I 0 0 1 0 (I 0 0 0 0 0 0 0 0 0
SP
0
0
0 0
0
0
0 0 0 0 0
1+
0 0 0
Notes: 0, no immunostaining; 1 t , 1% to 25% positive cells; 2 t , 26% to 50% positive cells; 3 t , 51 R’ to 75% positive cells: 4 t , >75% positive ~11s. Abbreviations: M, monophasic; B, biphasic; Ep. epithelial; Sp, spindle; KER. keratin; VIM, vimentin; NA, not available, * Ultrastructural studies performed. t Metastasis.
735
HUMAN PATHOLOGY
Volume 21, No. 7 (July 1990)
of gland1Jlar-like and solid patterns in epithelioid areas. The glandular-like FIGURE 1. (Left] Biphasic synovial sarcoma with a combination 1 3n of the same tumor showing keratin immunoreactivity restricted spaces contain proteinaceous material. (Right] lmmunostaining F to the epithelioid component.
lmmunocytochemistry
a single layer of flattened cells (Fig 2, left). In the remaining biphasic tumors, polygonal epithelial cells formed solid clusters. The spindle cell component of these biphasic tumors was made up of slender, fusiform cells with indistinct, tapering cytoplasm. The epithelial component was typically discrete where it formed glandular structures, but in a number of the tumors, particularly those with solid islands of epithelial cells, areas of apparent transition were observed. All the monophasic tumors were made up of densely packed spindle cells that varied in length but appeared shorter than normal fibroblasts. The cells were usually uniform and moderately plump with inconspicuous cytoplasm, and they were compactly grouped (Fig 3, left). There was scantly intercellular collagen in the most cellular areas, but sclerosis with foci of calcification was frequent. The cells were oriented within broad sheets forming bands and, on occasion, interlacing fascicles (Fig 3, right). In one tumor they formed a storiform pattern. In another, whorls of cells contained occasional squamoid areas intermixed with poorly developed glandular-like structures (Fig 4). In four tumors a hemangiopericytic pattern was present (Fig 5). Four monophasic tumors contained numbers of cells with a rhabdoid appearance.
The immunocytochemical results are summarized in Tables 2 and 3. The epithelial component of all 15 biphasic synovial sarcomas stained for keratin, but the spindle cells were positive in only six (Figs I, right and 2, right). Fifteen of the 24 monophasic tumors reacted for keratin (Fig 3, right). Two of the negative cases were recurrences in which previously resected specimens were positive for this marker. The keratin staining of the epithelial component of the tumor was usually diffuse. In contrast, reactivity in the spindle cells of both the biphasic and the monophasic tumors was almost invariably focal. The spindle cells of 20 of the 24 monophasic synovial sarcomas, and of every biphasic tumor, reacted for vimentin. Immunoreactivity for both vimentin and keratin occurred in the epithelial component of five of the 15 biphasic tumors, as well as in the 13 primary monophasic synovial sarcomas which originally stained for keratin. Coexpression of keratin and vimentin occurred not only in different groups of cells but also in the same individual cells. The percentage of cells which coexpressed both markers varied from tumor to tumor (Figs 5, top left and bottom right). In one monophasic tumor which contained 736
SYNOVIAL SARCOMA (Orddtiez et al]
FIGURE 2. [Left) Biphasic synovial sarcoma showing irregular crack Iirled by plump spindle cells. (Right] lmmunostaining preparation demonstrating keratin reactivity of the cells lining the crack-like spaces.
occurred throughout the cytoplasm, it tended to be stronger along the periphery of the spindle cells. All of these cases also reacted for keratin. Only six tumors (five biphasic and one monophasic) reacted for CEA. The number of positive cells staining with this marker was lower than those reacting for keratin or EMA. Fifteen primary monophasic (63%) and four metastatic (25%) spindle-cell tumors reacted for one of the epithelial markers. The marker most commonly detected was keratin, followed by EMA and CEA (Table 3). Thirteen of the 15 biphasic tumors, and two of the 24 monophasic tumors, reacted for UEAI. Staining in the epithelioid areas of these tumors was usually stronger along the periphery of the cells, and was most marked on the luminal surface of the cells lining glandular structures. Material within the lumens of some tumors also reacted for this lectin. Reactivity for Leu-7 occurred in six primary and Iive metastatic spindle-cell monophasic tumors and in the epithelial component of three biphasic sarcomas (Fig 5, bottom right). Immunostaining for S-100 protein occurred in two biphasic tumors and in one monophasic tumor in which reactivity was seen in both the primary tumor
ill-defined whorls of spindle cells, those at the center of the whorl stained strongly for keratin but were negative or weakly positive for vimentin, whereas the peripheral cells reacted strongly for the latter marker. Epithelial areas were rendered easily recognizable by combined keratin and vimentin immunostaining (Figs 4, top right and bottom left). Coexpression of both filaments was also seen in some of the cells located at the periphery of the nodules, indicating that a transition was occurring between the epithelioid and sarcomatoid components (Fig 4, bottom right). All four of the monophasic tumors which presented a. rhabdoid-like appearance reacted strongly for vimentin, but two also coexpressed keratin in a smaller Ipercentage of cells. Immunostaining for EMA was seen in the epithelial component of all biphasic tumors. In the gland-like structures, the staining occurred mainly along the apices of the cells and in the lumens. As a consequence of this staining pattern, lumens that were difficult to identify on routine light microscopy in the sollid tumors in hematoxylin-and-eosin sections could be visualized in the immunoperoxidase preparations. The spindle-cell component of five biphasic tumors reacted for EMA. Seven of the monophasic sarcomas stained for EMA and, although the staining 737
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Volume 21, No. 7 (July 1990)
FIGURE 3. [Left) Monophasic synovial sarcoma made of short, F)IUIw reacting for keratin.
spindle cells. [Right] Interlacing fascicles of fibroblast-like cells
timelanoma mors.
and the metastasis (case 32). The reaction involved both cytoplasm and nuclei and was usually focal (~30% of the cells); however one tumor tended to stain diffusely (80% of the cells, Fig 6). Although some variability in intensity of staining was seen between laminin and collagen IV, the reaction pattern appeared to be similar. In six biphasic tumors, continuous staining occurred along the epithelial areas. In most of the monophasic tumors, and in the spindle-cell component of the biphasic tumors, focal staining for collagen IV and/or laminin was detected between groups of cells. In the tumors with a hemangiopericytic pattern, unbroken staining occurred along the blood vessels enhancing the distinctive vasculature of the tumor (Fig 5, top left). On occasion, a significant amount of staining was visible around individual tumor cells. No immunoreactivity for any of the muscle markers, or for chromogranin A or the HMB-45 an-
antibody,
was obtained
in any of the tu-
Electron Microscopy In the biphasic tumors, cells of the epithelial component enclosing glandular lumina were connected at the luminal margins by tight junctions of moderate length, and below these junctions by a series of mature desmosomes with short tonoftlaments (Fig 7). Some infolding of lateral cell membranes was common, and filopodia protruded into narrow gaps between lateral cell surfaces. The epithelial cells rested on an intact basal lamina (Fig 8). Microvilli on the apical cell surfaces were often short and irregular in their distribution and length, but in many smaller lumina they were long and curved. Rare cilia were encountered on the epithelial cells and on an occasional spindle cell of three of the biphasic tumors (Fig
FIGURE 4. flop left) Biphasic synovial sarcoma presenting solid nodules of epithelioid areas containing poorly formed duct-like structures. flop right) Solid, well-demarcated epithelioid area reacting for keratin in the same tumor. (Bottom left) lmmunocytochemical preparation showing positive reactivity for vimentin in the sarcomatoid areas while the epithelioid component is negative. [Bottom right) lmmunostaining for vimentin preparation showing a poorly demarcated epithelioid area, suggesting a transition between the epithelioid and the sarcomatoid components of the tumor.
738
739
740
SYNOVIAL SARCOMA (Ord6Aez et al] FIGURE 5. (Top left) Laminin immunostaining accentuating the vascular pattern of a monophasic synovial sarcoma with hemangioperiq-tic pattern. (lop right) lmmunopreparation of the same tumor showing a strong paranuclear reaction for vimentin. (Bottom left) Cluster of cells stairling for keratin. (Bottom right] Cluster of spindle cells reacting for Leu 7.
7) as well as in six monophasic tumors. Some of the lumina contained amorphous material. Occasional small aggregates of glycogen were seen within the cytoplasm of the epithelial cells, but mucin was not found. Mitochondria occasionally filled the apical cytoplasm, lbut usually they were sparse and accompanied by only scattered slender cisternae. The cytoplasm of some cells contained intermediate filaments ranging from a few slender wisps (Fig 9) to diffuse aggregates. Although the filaments were focally condensed, myofilaments were never seen. Epithelial cells forming solid areas lacked tight junctions and microvilli. and had closely apposed cell surfaces with small but sometimes mature desmosomes and at most short tonofilaments. It was common to find small lumina amild apparently solid groups of cells (Fig 10). In the biphasic sarcomas, the spindle cells were usually loosely distributed within a collagenous stroma (Fig 11). but some neighboring cells were joined by small desmosomes or more primitive junctions. The cytoplasm tapered but did not branch. A few cytoplasmic lipid droplets were common, but organelles consisted primarily of a few cisternae that were som’etimes dilated and sparse mitochondria. Ther,e were some similarities between the spindle cells of the biphasic tumors and those of the monophasic sarcomas. Typically, the latter were short and fusiform with nonbranching cytoplasm. The cell surface was usually smooth (a few tumors had cells w-ith frequent filopodia), and intimate contact with neighboring spindle cells over part of a cell surface was the rule (Fig 12). At these sites, small cell attachments, either desmosomes or less structured junctions, were present in small numbers. Where cells were not in contact, slender zones of collagen intervened. These relationships between the cells were evident in longitudinal or oblique sections, and were well-demonstrated when the spindle cells were cut transversely (Fig 13). It was not possible to determine TABLE 3.
lmmunocytochemistry
whether the cells were in separate small groups, or were forming an interconnected meshwork. At some points, cells were separated by a wide band of collagen fibers with intermingled amorphous material similar in consistency and appearance to basal lamina. This material and collagen were abundant in the sclerosed areas of the tumor. A few cells had short, slender cytoplasmic extensions containing some nonspecific intermediate filaments but no myofilaments or arrays of microtubules. One monophasic tumor presented intracellular collagen in spindle tumor cells. Organelles in the monophasic sarcomas were similar m type and number to those within spindle cells in the biphasic tumors. The nuclei were frequently bland in appearance with smooth profiles and fine, evenly distributed chromatin. Nucleoli varied from small to moderate in size, and they were conspicuous against the background of dispersed chromatin. Chromatin clumping did occur in some tumors, and was common when preservation was less than optimal. In the four monophasic tumors containing rhabdoid cells, the globoid inclusions seen on light microscopy were formed by large aggregates of intermediate filaments (Fig 14). DISCUSSION The immunocytochemical findings from this study are in accord with those of previous reports. 1-5.2.5.p(iKeratin is the epithelial marker most often expressed and it can be detected in both the epithelial and spindle cell components. Immunostaining for keratin may, therefore, aid in differentiating a monophasic spindle-cell synovial sarcoma from softtissue neoplasms with similar histopathology. Epithelial membrane antigen and CEA are also expressed in some synovial sarcomas, but in significantlv fewer cases than those which react for keratin: this is parResults
in 39 Synovial
Sarcoma
Cases
Markers
Primary Tumors (n = 39) Biphasic (n = 15) Epithelial component
Keratin @)
EMA 6)
CXA (9%)
15
15 (100)
.i (33) 0
(100) Spindle Monophasic
(Xls (n = 24)
Distant Metastases (n = 19) Biphasic (n = 3) Epithelial component
6
(‘w
(9;)
15 (63)
7 (29)
(4)
3
0
3
( 100) Spindle
cells
2 (67)
Monophasic
(n = 16)
I
UEAl (%)
13
1 (33) 9 (13)
0
4, 12
(“3
(X0)
2 (8)
20 (83)
3
2 (67) 3 (100) 13 (81)
0
741
2 (671 2 (13)
S-100
(8)
1
(87) 4
( 100)
( 100)
Vimentin (s7r)
(7) (1:) 1 (4)
0 0
1 Cf.9
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Volume 21, No. 7 (July 1990)
FIGURE 6. [Left) Biphasic svnovial sarcoma demonstrating reactivity for S-100 in the epithelioid cells. [Right) Monophasic tumor showing numerous s&dle dells staining for S-100.
in the intensity of the staining correlated with the quantity of intermediate filaments seen by electron microscopy. Since vimentin can also be found in a wide variety of epithelial neoplasms, reactivity for this marker alone has little diagnostic value. However, when coexpressed with other intermediate filaments, it may facilitate the identification of certain neoplasms. An example is the renal rhabdoid tumor,*’ in which the eosinophilic cells contain prominent globular, hyaline inclusions that impart a rhabdomyoblast-like appearance. Ultrastructurally, the inclusions are accumulations of intermediate filaments, and they react for vimentin and keratin but seldom for muscle markers.28~2g So-called malignant rhabdoid tumors have been reported in a variety of extrarenal sites including soft tissues, but Tsuneyoshi et a130 were able to identify rhabdoid cells in a variety of specific soft-tissue sarcomas including epithelioid sarcomas, extraskeletal myxoid chondrosarcomas, and synovial sarcomas. It seems likely that extrarenal malignant rhabdoid tumors are a heterogeneous group with only a morphologic resemblance to the renal neoplasm. All four of the tumors with rhabdoid features in our series coexpressed vimentin and keratin, and the staining was concentrated in the cytoplasmic inclusions. Ultrastructurally, the intermediate filaments in
titularly the case with the monophasic tumors. In our study, EMA immunostaining occurred in seven of 24 primary monophasic tumors and in two of 16 metastatic monophasic tumors. Although all of our cases that stained for EMA also reacted for keratin, EMApositive, keratin-negative monophasic tumors have been reported.’ Therefore, EMA staining should be performed on suspected synovial sarcomas when keratin staining is not contributory. Only six of the synovial sarcomas in our series reacted for CEA, and the staining was focal and confined, in five of the cases, to the epithelial elements of the biphasic tumors, a confirmation of earlier observations.‘.* Corson et al* found reactivity for CEA in three of eight biphasic tumors, localized in the epithelial component, and in only one of 16 monophasic tumors. As was the case in our series, all the tumors in their study that stained for CEA also reacted for keratin. Based on these observations, we believe that CEA has limited value in the diagnosis of synovial sarcoma. Since the tumors in our series that reacted for EMA and CEA also stained for keratin, the last appears to be the epithelial marker of choice. Reactivity for vimentin was obtained in the spindle cells of all the tumors in our series, both biphasic and monophasic. The epithelial cells also stained for vimentin in five of the 15 biphasic tumors. Variations 742
SYNOVIAL SARCOMA (Ord6riez et al)
FIGURE 7. A small lumen from a biphasic synovial sarcoma contains irregular. short microvilli and occasional cilia. The tight junctions and desmosomes are evident. (Magnification x 21,000.)
Recent studies have indicated that antibodies to Leu-7 can recognize an antigenic constituent of myelin-associated glycoprotein and they may. therefore, have some potential in separating neurogenic sarcoma from other spindle-cell sarcomas, including monophasic synovial sarcoma.““.q’l That we were able to identify reactivity for Leu-7 in 11 synovial sarcomas confirms that this marker cannot reliably separate a monophasic synovial sarcoma from a Schwann cell tumor.fi S- 100 protein is known to be expressed in a number of malignant schwannomas,4i-“” while synovial sarcomas have been reported to be negative for this marker.1”4 It has been suggested that staining for S- 100 could be used to distinguish monophasic and biphasic synovial sarcomas from malignant Schwann cell tumors, including those with glandular differentiation.1,“4 This argument cannot be sustained in the light of our observation of S-100 immunoreactivity in both the epithelial and the spindle-cell elements of some synovial sarcomas. Collagen IV and laminin are two major components of the basal lamina that can be demonstrated by immunocytochemical methods, and it has been reported that the immunostaining pattern for laminin can help in the diagnosis of certain soft-tissue tumors.45 We were able to demonstrate continuous reactivity for collagen IV and laminin along the epithelial component of biphasic tumors, and the immunostaining of these substances was helpful in revealing and defining the extent of epithelial areas that were
the inclusions did not display specific features, although they occasionally condensed into bundles resembling the cytokeratin of squamous cell carcinomas. Inclusions composed of intermediate filaments have been reported in a variety of epithelial”‘-33 and mesenchylmal’ ‘zN~’ tumors, as well as in melanomas,‘15 meningiomas,:‘” and mesotheliomas.:” Even nonspecific filaments may contribute to the diagnosis of a tumor in which they occur frequently, notably neuroendoepithelioicl sarcomas’ l and cutaneous crine (Merkel cell) carcinomas.31,““.3s The inclusions may also serve to distinguish monophasic synovial sarcoma from hemangiopericytoma. It has been our experience that the latter does not express keratin or possess large accumulations of intermediate filaments. Synovial sarcoma can potentially be confused with a nulmber of other defined sarcomas, particularly with leiomyosarcoma, neurogenic sarcoma, fibrosarcoma, and malignant fibrous histiocytoma. While a positive reaction for keratin may offer support for a diagnosis of synovial sarcoma, its contribution is diminished when leiomyosarcoma is included in the differential diagnosis, since some leiomyosarcomas have been reported to stain for keratin and EMA.12 Therefore, immunostaining for muscle markers such as smooth-muscle actin or desmin 12,13 The two antibodies to should also be performed. actin used in our project react strongly in paraffinembedded tissue and are more sensitive than desmin in the diagnosis of leiomyosarcomas. 743
HUh4AN PATHOLOGY
Volume 21, No. 7 (July 1990)
FIGURE 8. The epifhelial rest on an intact
Cells
X 7,000.)
x 9,500.)
SYNOVIAL SARCOMA (Ord6riez et al]
FIGURE IO. Narrow lumina within a solid area of epithelial cells. (Magnification x 4,500.)
obscure in hematoxvlin-and-eosin preparations. Immunostaining for collagen IV and laminin in the spindle cells of the biphasic and the monophasic tumors was limited to small foci, and usually correlated with the observation of scanty basal lamina by electron microscopy. The reaction contrasted markedly with the diffuse staining for laminin surrounding individual ‘cells that has been reported in leiomyosarcomas and in some Schwann cell tumors.q” In our study, the demonstration of keratin immunoreactivity often facilitated the diagnosis of monophasic synovial sarcoma, but there remained a significant number of tumors (37% of the primary and 75% of the metastatic monophasic synovial sarc-omas) t!hat failed to express epithelial markers or, with the exception of vimentin, any of the other markers used in the study. Various reasons can be suggested. The marker may have been lost, as probably occurred in six cases of monophasic spindle-cell tumors in which none could be demonstrated in the recurreme or metastasis (Table 2). It is also possible that variation in the sample could account for the staining being focal and confined to a very small number of cells. In thle differential diagnosis of monophasic synovial sarcoma, separation from a primitive, unclassified sarcoma may not be possible, but defined sarcomas with comparable histopathology can usually be identified from their combined light- and electronmicroscopic features.
Compared with the cells of a fibrosarcoma, the spindle cells of a monophasic synovial sarcoma are shorter, and their cytoplasm tapers more sharply and does not branch. Neoplastic fibroblasts do not generally show such consistent close contact of neighboring cell membranes within small clusters of intimately apposed cells, nor do they possess basal lamina. Where collagen intervenes between cells in a monophasic synovial sarcoma, it frequently forms delicate partitions. The cytoplasm tends to have fewer organelles, especially rough endoplasmic reticulum, than ftbrosarcoma cells, and myofibroblastic differentiation is unique to true fibroblastic tumors. ‘The nuclei in monophasic synovial sarcomas are characteristically oval with smooth profiles and fine, evenly dispersed chromatin. Malignant fibrous histiocytoma cells have organelles similar to those in fibrosarcoma, although the cytoplastn is often more extensive and it may contain diffuse zones of intermediate filaments. Intracytoplasmic collagen, which was present in one of our cases, has been reported in various types of sarcomas, including malignant fibrous histiocytomas-l” There are some similarities between monophasic synovial sarcoma and stromal sarcomas of uterus and breast, but the clinical findings usually prevent confusion. Nerve sheath sarcomas display a broad spectrum of histopathology, but ultrastructurally the cells often have long cytoplasmic extensions of uniform caliber that contain filaments and microtubules, and mesaxon formation may be found.+7,AS The spindle745
HUMAN PATHOLOGY
Volume 21. No. 7 (JulY 1990)
FIGURE 11. Biphasic synovial sarcoma in which there is o clear demarcation between the epithelial cells forming the glandular structure and the adjacent spindle cells, many of which contain lipid droplets, (Magnification x 2,800.)
FIGURE 12. The plane of section passes obliquely through spindle ceils of a monophasic synovial sarcoma. Some cells are in direct contact with their neighbors, while others are separated by slender bands of collagen, (Magnification x 4,200.)
746
SYNOVIAL SARCOMA (Ord6riez et al]
FIGURE 13. Cells of the same tumor seen in Figure 6 sectioned transversely. Intimate apposition of cells within tiny bundles is highlighted by the delicate collagen partitions. (Magnification )i 4,200.)
cell component of the rare malignant schwannoma with epithelial differentiation has schwannian features. Malignant melanoma forming in soft tissues, formerly termed clear-cell sarcoma, can resemble a monophasic synovial sarcoma, but the cells are often rounder, nucleoli are typically large and dense, premelanosomes are present, and spindle-cell tumors may manifest some neural differentiation.-‘!’ The histogenesis of synovial sarcoma remains an intriguing question. For many years, the tumors were believed to originate from synovial cells, but when ultrastructural studies failed to show obvious synovial features in the tumors,5”.“’ doubt was cast on this theory. The immunocytochemical evidence does not indicate a histogenetic relationship between the tumors and synovial lining cells. Synovial cells do not express epithelial markers or bind UEAI lectin, whereas synovial sarcomas may do both.‘.*” Since synovial sarcomas express epithelial markers such as keratin, EMA, and CEA, and possess well-developed desmosomes which have been reported to react to desmosomal proteins exclusively associated with epithelial cell~.~~ some investigators have proposed that synovial sarcoma is a unique form of carcinosarcoma confined1 to soft tissues.25.5” Genuine carcinosarcomas are uncommon tumors, often difficult to diagnose with certainty, although some may show specific differentiation m the sarcomatous component, as in the leiomyosarcomatous portion of renal-cell carcinosarcomas.“” It is becoming increasingly recognized that there is not always a rigid separation of epithelial from mes-
enchymal cells. Morphologic distinctions between the two are often blurred. We have. for example, been impressed with the ultrastructural resemblance of sarcomatoid squamous carcinoma cells to fibroblasts. The myoepithelial cell is a hybrid with epithelial and smooth-muscle features which can be variably expressed in neoplasms. Transitions from an epithelial to a mesenchymal phenotype are well-documented in vertebrate development, and are observed in in vitro models.“” Gene expression can be modified by environmental conditions and lead to profound alterations in cell structure: mouse sarcoma cells transfected with complementary DNA encoding chick LCAM assume an epithelial morphology.“” A genetic abnormality in synovial sarcoma has been reported on a number of occasions.5T In spite of light-microscopic similarities between biphasic synovial sarcomas and the synovial membrane, it has not been established that the tumors are displaying authentic synovial differentiation.“*38 The disparate anatomic locations of the primary tumors, often far removed from the nearest articular surface, and the infrequency with which a joint surface is clearly involved, are evidence against sarcomas being truly synovial. The synovial lining is composed of thinly stratified and looselv grouped cells with slender cytoplasmic extensions that arborize over the surface, and desmosomes are notably absent.:” When the tumor cells are compared with those of the normal synovial lining (tendon sheath appears to have a similar construction), there are no obvious resemblances between the two. An analogy with mesothelioma can be seen. Ex747
HUMAN PATHOLOGY
Volume 21, No. 7 (July 1990)
FIGURE 14 A diffuse aggregate of intermediate filaments displaces organelles to the peripheral cytoplasm giving the cell a rhabdoid appearance on light microscopy. (Magnification x 23,000.)
perimental studies have shown that the subserosal spindle cell responds to injury of the overlying epithelium by proliferating, enlarging, assuming first low-molecular weight then high-molecular weight keratin immunoreactivity, and repairing breaches in the mesothelial surface.5Y.6” These observations have led to the postulate that the spectrum of histopathology displayed by mesotheliomas can be accounted for by varied differentiation of the submesothelial spindle cell towards fibroblasts, epithelial (mesothelial) cells, or intermediate forms that lack the extensive endoplasmic reticulum of the mature fibroblasts or myofilaments of a myofibroblast, and also do not possess the basal lamina, complex cell attachments, or luxurious microvilli of the surface cells. The occurrence of the uncommon mixed variant of mesothelioma, in which spaces lined by a single layer of epithelium are interspersed throughout a background of spindle cells, can be explained by this hypothesis. In like manner, a synovial sarcoma could be postulated to develop from a primitive mesenchymal with the inherent or acquired ability to form surface epithelium.
a combined peroxidase-antiperoxidase/avidin-biotin-peroxidase complex procedure. HUM PATHOL 17:1107-l 115, 1986 2. Corson JM, Weiss LM, Banks-Schlegel SP. et al: Keratin proteins and carcinoembryonic antigen in synovial sarcomas: An immunohistochemical study of 24 cases. HUM PATHOL 15:615-621, 1984 3. Carson JM, Weiss LM, Banks-Schlegel SP, et al: Keratin proteins in synovial sarcoma. Am J Surg Pathol 7:107-109, 1983 4. Fisher C: Synovial sarcoma: Ultrastructural and immunohistochemical features of epithelial differentiation in monophasic and biphasic tumors. HUM PATHOL 17:996-1008, 1986 5. Miettinen M, Veli-Pekka L, Virtanen I: Monophasic synovial sarcoma of spindle-cell type. Epithelial differentiation as revealed by ultrastructural features, content of prekeratin and binding of peanut agglutinin. Virchows Arch [B] 44: 187-199, 1983 6. Swanson PE. Manivel IC. Wick MR: Immunoreactivitv for Leu-7 in neurofibrosarcoma and’other spindle cell sarcomas of soft tissue. Am J Pathol 126:546-560, 1987 7. Krall RA, Kostianovsky M, Patchefsky AS: Synovial sarcoma. A clinical, pathological, and ultrastructural study of 26 cases supporting the recognition of a monophasic variant. Am J Surg Pathol 5:137-151, 1981 8. Alvarez-Fernandez E, Escalona-Zapata J: Monophasic mesenchymal synovial sarcoma: Its identification by tissue culture. Cancer 47:628-635, 1981 9. Daimaru Y, Hashimoto H, Tsuneyoshi M, et al: Epithelial profile of epithelioid sarcoma. An immunohistochemical analysis of eight cases. Cancer 59:134-141, 1987 10. Manivel JC, Wick MR, Dehner LP, et al: Epithelioid sarcoma. An immunohistochemical study. Am J Clin Pathol 87:319326, 1987 11. Meis JM, Mackay B, Ord6tiez NC: Epithelioid sarcoma: An immunohistochemical and ultrastructural study. Surg Pathol 1:13-31, 1988 12. Miettinen M: Immunoreactivity for cytokeratin and epithelial membrane antigen in leiomyosarcoma. Arch Pathol Lab Med 112:637-640, 1988
Acknowledgment. The authors wish to thank Thomas E. Brooks, HT, Hazel Dalton, HT, Sharon Thompson, HT. and Elsa Ramos, HT, for their technical assistance.
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JC. Swanson PE, et al: Synovial sarand immunohistochemical analysis by
748
SYNOVIAL SARCOMA (Ord6riez
13. Norton AJ, Thomas JA, Isaacson PG: Cytokerdtin-specific monoclonal antibodies are reactive with tumours of smooth muscle derivation. An immunocytochemical and biochemical study using antibodies to intermediate filament cytoskeletal proteins. Histopathology 11:487-4!)9, 1987 14. Coidre J-M, De Mascarel A. Trojan MM,et al: Immunohistochemical study of rhabdomyosarcoma. Unexpected staining with S-100 protein and cytokeratin. J Pathol 155: 127-132. I988 1.5. Miettinen M, Rapola J: Immunohistochemical spectrum of rhabdomvosarcoma and rhabdomyosarcoma-like tumors. Expression of cytokeratin and 6X-KD neurofilament keratins. Am J Surg I’dthol 13:120-132. 1989 16. WlGss SW, Bratthauer GL, Morris PA: Postradiation mahgnant fibrous histiocytoma expressing cytokeratin. Implications lor the immunodiagnosis of sarcoma. Am J Surg Pathol 12:554.55X. I988 17. Grav M. Rosenberg .4, Bhan A. et al: Cytokeratin expression bv ephthelioid vascular neoplasms. Lab Invest 60:34A. 1989 (abstr) IX. Enzinger FM. Weiss SW: Synovial sarcoma, in Soft Tissue ‘l‘umors. St Louis, MO. Mosby. 1988, pp 659-688 19. Evans H: Svnovial sarcoma. A study of 23 biphasic and 17 probably monophasic examples. Pathol Annu 2:309-33 1, 1980 20. Hajdu SI. Shiu MH. Fortner JG: Tendosynovial sarcoma. .\ clinicopathological stud, of 136 cases. Cancer 39: 1201-1217. 1977 2 I Ord6Aez NC;.Batsakis JG: Comparisons of c’lrx ~uropaeu\ I lectin and factor VIII-related antigen in vascular lesions. Arch Pathol Lab Med 10X:129-132. 1984 22. Ordbiier IiG. Ziaolong J, Hickey R: Comparison of HMB45 monoclonal antibody and S-100 protein in the immunohistochemical diagnosis of melanoma. Am .J Clin Pathol 90:385-390, 19X8 23. Hsu S-M. Raine L, Fanger H: Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580, 1981 24. Ord6iiez NC;. Manning J, Brooks T: Effect of trypsinizarion on the immunostaining of formalin-fixed. paraffin-embedded rissues. Am J Surg Pathol 12:121-329. I988 25, Miettinen M, \ lrtanen I: Synovial sarcoma--:\ misnomer. * .\m J Pathol I1 /: IX- 2 5”3 1984 26. Salisbury JR, Isaacson PC;: Synovial sarcoma: An immunohistochemical study. J Pathol 147:49-57. 1985 27. Sotelo-Avila
