Malignant Peripheral Nerve Sheath Tumors: An lmmunohistochemical Ultrastructural Features
Study in Relation to
TAKANORI HIROSE, MD, TADASHI HASEGAWA, KUNIHIKO SEKI, MD, TOSHIAKI SANO, MD, AND KAZUO HIZAWA, MD The constituent cells in malignant peripheral nerve sheath tumors were examined by studying the expression of immunohistochemical markers for Schwann cells and perineurial cells in relation to ultrastructural features in 12 malignant peripheral nerve sheath tumors. Ultrastructural studies demonstrated mixed proliferation of Schwann cells, perineurial cells, fibroblastic cells, and primitive cells in many malignant peripheral nerve sheath tumors. Expression of S-100 protein was well correlated with Schwann cell-like differentiation of tumor cells. However, Leu-7 and epithelial membrane antigen, which have been considered to be specific to Schwann cells and perineurial cells, respectively, were common to Schwann cells, perineurial cells, and primitive cells. The common immunophenotypic expression suggests a close relationship among these cell types. The unusual expression of cytokeratin could be explained by the plasticity of intermediate filament expression. HUM PATHOL 23:865-870. Copyright 0 1992 by W.B. Saunders Company
Malignant peripheral nerve sheath tumors (MPNSTs) are poorly defined sarcomas and show great histologic variation.’ Ultrastructural and immunohistochemical studies are necessary for accurate diagnosis of this type of tumor, unless the tumor is associated with neurofihromatosis type 1 or has continuity with a nerve. Previous ultrastructural studies have indicated a Schwann cell nature of the cells in MPNSTs.“.’ These tumors have been found to contain other cell types derived from the peripheral nerve sheath as well, such as heterperineurial cells7-” We also have demonstrated ogeneous cell populations in six MPNSTs by S-l 00 protein-immunostaining and ultrastructural studies.‘” Recently, there have been many immunohistochemical S-100 protein, Leu-7, myelin studies of MPNSTs.“-‘* basic protein, and glial fibrillary acidic protein (GFAP) have been considered to be immunohistochemical markers of Schwann cells in the peripheral nervous system.‘” E P ithelial membrane antigen (EMA) has been demonstrated to be localized in the perineurium of peripheral nerves and has been used as a perineurial cell marker in studies of benign peripheral nerve sheath tuan mors (RPNSTS).~~-~’ We previously have reported MPNST showing diffuse EMA immunoreactivity.” In the present study we examined the irnmunohistochem-
MD, EIJI KUDO, MD,
ical expression of these markers of Schwann cells and perineurial cells as well as some cytoskeletal proteins in 12 tumors, and correlated the results with the ultrastructural features of the tumors.
MATERIALS
AND METHODS
Twelve tumors with characteristic ultrastructural features of MPNSTS’~,‘~ were selected for this study. We have reported the ultrastructural features of seven of these tumors (Table 1; cases no. 2, 3, 6, 7, 9, 10, and 11).‘“,23 Three patients (cases no. 1, 3, and 11) had neurofibromatosis type 1 and in three patients (cases no. 3, 6, and 9) the tumors had continuity with nerves. For immunohistochemical studies, in addition to hematoxylin-eosin staining, 4 pm-thick sections from specimens fixed in 10% formalin and embedded in paraffin were prepared. Immunohistochemical staining was performed using the avidinbiotin-peroxidase complex method with an ABC kit (Vector Laboratories, Burlingame, CA). The primary antibodies used are shown in Table 1. Polyclonal anti-S-l 00 protein antibody (diluted 1:2,000; Takahashi et alz6) and monoclonal anti-leu7 antibody (diluted 1:50; Becton Dickinson, Mountain View, CA) were used as Schwann cell markers, and monoclonal antiEMA antibody (diluted 1:50; DAKOPATTS a/s, Glostrup, Denmark) was used as a perineurial marker. To study the cytoskeletal proteins of MPNSTs we used monoclonal anti-olsmooth muscle actin (diluted 1: 1,000; BioMakor, Rehovot, Israel) and vimentin (diluted 1: 10; DAKOPATIS) antibodies, as well as polyclonal anti-GFAP (diluted 1:500; DAKOPATTS) and neurofilament (200 kd, diluted 1: 100; our laboratory) antibodies. For the study of cytokeratin five different monoclonal antibodies were used: PKKl (44, 46, 52, and 54 kd, diluted 1:lOO; Labsystems, Helsinki, Finland), CAM 5.2 (39 and 43 kd, diluted 1: 1; Becton Dickinson), 35PH 11 (54 kd, diluted 1: 1,000; Enzo, New York, NY), 34PE12 (57 and 66 kd, diluted l:l,OOO; Enzo), and 34PB4 (68 kd, diluted l:l,OOO; Enzo). External lamina was examined with polyclonal anti-type IV collagen antibody (diluted 1:200; Advance, Tokyo, Japan). Immunostaining with anticytokeratin and anti-type IV collagen antibodies required predigestion of the sections with trypsin. For electron microscopic studies specimens of primary, recurrent, or metastatic tumors obtained at surgery were cut into small cubes and fixed in 3% glutaraldehyde in phosphate buffer. The specimens were then washed with buffer solution, postfixed in 1% osmium tetroxide in phosphate buffer, dehydrated, and embedded in Epon 812. For correlation with immunohistochemical findings blocks containing areas that were microscopically similar to immunohistochemical sections were selected. Thin sections from two to nine blocks (mean, 5.8 blocks) of each tumor were stained with uranyl acetate and lead citrate, and examined with an electron microscope. We correlated neoplastic cells of each tumor with the cells
From the First Department of Pathology, University of Tokushima School of Medicine, Tokushima, Japan. Accepted for publication September 30, 1091. Key umrd~: peripheral nerve sheath tumor, malignant, ultrastructure, imnlunohistochemistry. Address correspondence and reprint requests to Takanori Hirose, MD, The First Department of Pathology, University of Tokushima School of Medicine, Kuramoto-cho 3, Tokushima 770, Japan. Copyright C 1992 by W.B. Saunders Company 00468177/92/2308-0005$500/O
865
HUMAN PATHOLOGY
TABLE 1.
Clinical and lmmunohistochemical
Volume 23, No. 8 (August
1992)
Features of Malignant Peripheral Nerve Sheath Tumors Cytokeratin
Case
Age (yr)/ Sex
Location of Tumor
SlOO
Leu-7
EMA
VIM
CAM
PKK
1 2
46/F 75/M
R upper arm R scapular
++ ++
+ ~
+ ~
++ ++
-
_
3
30/F
+
_
~
++
+
+
+
4 5 6 7 8 9
55/M 63/F 37/M 52/M 65/F 20/F
+ ++ _
_
+
+ _ _
++
+ + -
_ _ _ _
_
++ + ++ t+ ++ ++
_
~ + _
_
_
10 11 12
23/F 60/F 58/M
L brachial plexus Prostate L chest wall R chest wall L back R chest wall R brachial plexus I, elbow Ankle L back
_ _ _
+
t
_
t _ _
+
_ _
_ _
5
4
5
11
3
No.
region
Total
_ ++ ~ +
Symbols: ++, dilhtsely positive; +, focally positive; -, negative. Abbreviations: EMA, epithelial membrane antigen: VIM. vimentin; ASMA. a-smooth muscle actin.
constituting normal peripheral nerve sheath and BPNSTs: Schwann cells, perineurial cells, and fibroblasts.“5~27 RESULTS Light Microscopic
34/3B4
GFAP
NF200
ASMA
Type 4 Collagen
_ _
_ _
_ _
_
+ +
+
_
_
_
_
+
_ _
_ _
_ _
_ _
_ _ _ _ _ -
_ _ _ _ _ _
_ _ _ _ _
_ _ _
_ _ _
_ _ _
_ _ _
_ _ -
_ _ _
+ _
1
2
1
0
0
0
0
10
GFAP,
glial fibrillary
35PEll
34PE12
_
acidic
protein;
NF200,
neurofilament
_ t t + + +
+
200KD;
1, center). In case no. 7 spindle-shaped cells were arranged in storiform and whorl patterns.*’ Five tumors (cases no. 8, 9, 10, 11, and 12) consisted entirely of poorly differentiated, short, spindle-shaped cells showing a vague fascicular pattern (Fig 1, right).
Findings
All tumors were principally composed of spindle tumor cells arranged in intersecting fascicles, but there was great histologic variation. Small parts of three tumors (cases no. 1,2, and 3) had neurofibroma-like areas in which spindle cells with wavy nuclei grew irregularly in fibromyxoid stroma (Fig 1, left). In case no. 1 there were many cellular bundles surrounded by perineuriumlike whorls. Nuclear palisading and epithelioid tumor cells were noted in cases no. 4 and 5, respectively (Fig
lmmunohistochemical Findings and Related Ultrastructural Features The results of immunostaining of the 12 MPNSTs are shown in Table 1. S-l 00 protein immunoreactivity was demonstrated in five tumors (cases no. 1, 2, 3, 4, and 5). Many wavy spindle cells in neurofibroma-like areas of cases no. 1, 2, and 3 were stained, whereas poorly differentiated short-spindle cells were not. In neurofibroma-like areas electron microscopy disclosed
FIGURE 1. (Left) A neurofibroma-like area of case no. 3 showing an irregular growth of wavy spindle cells in ftbromyxoid stroma. (Magnification X 174.) (Center) Case 4. An MPNST showing well-developed nuclear palisading. (Magnification x174.) (Right) Case no. 9. Fascicular growth of short, spindle-shaped tumor cells (Magnification x 174.)
MALIGNANT
PERIPHERAL NERVE SHEATH TUMORS
mixed proliferation of Schwann cells, perineurial cells, and fibroblastic cells. Schwann cells, covered by distinctive external laminas (Fig 3a), had broad and occasionally branching cell processes and numerous intermediate filaments. Many S-l 00 protein-positive spindle cells were diffusely distributed in cases no. 4 and 5 (Fig 2a), both of which were predominantly composed of Schwann cell-like cells with numerous cytoplasmic processes entangled and connected with one another (Fig 3b). Leu-7 immunoreactivity was often detected in areas of poorly differentiated, short, spindle cells (cases no. 1, 9, and 10; Fig 2b). Ultrastructural examination of these areas showed that the compact tumor cells had a few short cell processes with no interdigitation and were often connected by primitive junctions. Electron-dense material was seen in the intercellular space (Fig 3~). Because these short, spindle-shaped tumor cells showed no apparent differentiation into Schwann cells or peri-
FIGURE 2. Immunohistostainings of chemical MPNSTs. (a) Case no. 4. Many S-IdO’protein-positive tumor cells. (Magnification x129.)(b) Case 9. Membranous immunoreactivity for Leu-7 on numerous primitive tumor cells. (Magnification X344.) (c) Case no. I. Perineurium-like cellular whorls showing weak reactivity for EMA. (Magnification x172.) (d) Case no. 10. Fine, granular immunoreactions for EMA localized in cell membranes of many primitive tumor cells. (Magnification x258.) (e) Case no. 5. Many scattered tumor cells with cytokeratin (CAM 5.2)-reactive cytoplasm. (Magnitication ~258.) (f) Case no. 3. A few plump tumor cells showing high molecular weight cytokeratin (34pE12) immunoreactivity. (Magnification x172.)
(Hirose et al)
neurial cells, the tumor cells were designated as primitive cells. Some spindle-shaped cells of the cellular fascicles of case no. 6 also showed Leu-7 immunoreactivity; this tumor consisted of perineurial-like cells with long, slender cell processes covered by well-developed external laminas and some pinocytotic vesicles along the cell membrane (Fig 3d). Epithelial membrane antigen-positive tumor cells were present in five tumors. In case no. 1 reaction products were localized in cellular whorls resembling the perineurial sheath found in neurofibroma-like areas (Fig 2c) and these cells showed ultrastructural characteristics of perineurial cells (Fig 3e). Small aggregates of primitive cells (cases no. 9 and 10; Fig 2d) and spindle-shaped cells (case no. 4), which were ultrastructurally similar to Schwann cells (Fig 3b), showed reactivity along their cell membranes, whereas the tumor cells of case no. 7 showed diffuse reactivity. The ultrastructural features of the cells in case no. 7 seemed to
HUMAN PATHOLOGY
Volume 23, No. 8 (August
1992)
FIGURE 3. (a) Case no. 3. Mixed proliferation of a Schwann cell (S) covered by a continuous external lamina and ftbroblastic ceils (F) with well-developed rough endoplasmic reticulum. The arrow indicates long-spacing collagen. (Magnification x6.240.) (b) Case no. 4. Closely packed Schwann cell-like tumor cells with numerous interdigitating cell processes. (Magnification x7.800.) (c) Case no. 9. An ultramicrograph of primitive tumor cells, occasionally connected by primitive junctions (arrowhead). Note electron-dense substances in the intercellular space. (Magnification ~7,800.) (d) Case no. 6. Perineurial-like cells with slender cell processes covered by well-developed external laminas. A few pinocytotic vesicles (arrowheads) are seen along the cell membrane. (Magnification x 13,260.) (e) Case no. I. Perineurial tumor cells characterized by long, slender cell processes, pinocytotic vesicles, and external laminas. (Magnification x13,260.)
lial differentiation was detected in any tumor ultrastructurally. No tumor reacted with anti-cu-smooth muscle actin antibody, which showed intense immunoreaction in vascular smooth muscle cells. Linear, pericellular reactions for type IV collagen were demonstrated in 10 tumors.
be intermediate between those of perineurial and Schwann cells.“’ Vimentin reactivity was observed in all tumors except that in case no. 11, but no reactivity for GFAP or neurofilament 200 kd (both of which were localized in nonneoplastic nerve fibers in the tumors) was detected in any tumor. A few cytokeratin-positive cells were unexpectedly detected in three tumors (cases no. 3, 5, and 6) stained with CAM 5.2. The positive cells, which were spindle-shaped or epithelioid, were scattered among negative cells and were not arranged in any epithelial pattern, such as a nest or tubule (Fig 2e). Other anticytokeratin antibodies (35PH 11, 34PE12, and PKKl) also reacted with a few tumor cells in two, one, and one tumors (Fig 2I), respectively, whereas no immunoreaction with 34PB4 was demonstrated. No definite epithe-
DISCUSSION Because they have no specific microscopic features and show great histologic variation,’ MPNSTs are difficult to differentiate from other soft tissue tumors. As found in the present study, the microscopic features of MPNSTs varied from well-differentiated areas with prominent palisading and neurofibroma-like patterns 868
MALIGNANT
PERIPHERAL NERVE SHEATH TUMORS
(Hirose et al)
tumor. We also did not detect actin in any tumor. Actin is present in almost all mammalian cells, where it exists as one of at least six isoforms that show tissue specificity.3’ Thus, it seems necessary to further investigate actin expression using monoclonal antibodies specific for each isoform. Miettinen” also used monoclonal antibodies specific to muscular actin isoforms. Our findings indicate that MPNSTs do not seem to have muscle-type actins, although they may have other isoforms. Differences in actin isoforms may be of practical use in differentiating MPNSTs from myogenic tumors. Some groups have reported a few MPNSTs showing reactivity for cytokeratin.‘7,28,32 In the present study cytokeratin immunoreactivity was demonstrated in three of 12 tumors studied. These three tumors were diagnosed as MPNSTs from their ultrastructural features and immunoreactivity for S-100 protein and Leu-7. Their unusual expression of cytokeratin might be explained by epithelial differentiation of tumor cells, as suggested by other investigators.32 Malignant peripheral nerve sheath tumors have been shown occasionally to contain epithelial elements with well-develo ed glands and epithelial nests as heterotopic elements P; however, the cytokeratin-positive cells found in the present study were spindle-shaped or polygonal and did not show any epithelial structures microscopically or ultrastructurally. Moreover, none of the three cytokeratin-positive tumors showed reactivity for EMA. Recently, Ortonne et al33 reported cytokeratin immunoreactivity in lamellar cells of sensory receptors and perineurial cells of pig skin. If similar expression occurs in humans, cytokeratin reactivity might be regarded as indicating perineurial cell differentiation of tumor cells. However, the normal perineurium included in the sections examined in the present study did not react with monoclonal anti-cytokeratin antibodies. Another explanation for the presence of cytokeratin, which seems to be the most reasonable, is the plasticity of intermediate filament expression.34s35 The expression of intermediate filaments has been thought to be relatively specific and stable for given cell types, but many cases of unusual or unexpected expression of intermediate filaments in normal, reactive, and neoplastic tissues have been reported. Cytokeratin expression has been observed in many fetal and adult mesenchymal cells, such as endothelial cells and smooth and striated muscle cells,“4s36 as well as in many types of sarcomas, such as synovial sarcoma,37 epithelioid sarcoma,38 leiomyosarcoma,3” rhabdomyosarcoma,40 Ewing’s sarcoma,41 malignant fibrous histiocytoma,42 and epithelioid angiosarcoma.43 In sarcomas the presence of a few cytokeratin-positive tumor cells might be interpreted as a nonspecific shift of intermediate filaments induced by some unknown regulatory mechanisms connected with intermediate filament expression. Ultrastructurally, MPNSTs seem to be composed of Schwann cells, perineurial cells, and primitive cells in varying proportions. The immunohistochemical expression of Leu-7 and EMA, which is considered to be specific to Schwann cells and perineurial cells, respectively, was common to Schwann cells, perineurial cells, and primitive cells. The common immunopheno-
to poorly differentiated areas containing only diffusely distributed primitive cells. These different growth patterns were often intermingled in the same tumor. The ultrastructural features of MPNSTs also vary greatly. Some previous studies have indicated fine structural similarities between the cells of MPNSTs and Schwann cells,‘” whereas other studies have indicated the presence of other cell types as well.7-g We also reported the presence of perineurial cells, fibroblastic cells, and primitive cells (as well as Schwann cells) in MPNSTs.” In the present study we examined the correlation of ultrastructural findings with the expressions of immunohistochemical markers. S-100 protein, which is thought to be restricted to Schwann cells in normal peripheral nerves and neurofibromas,“,” was detected in five tumors in which Schwann cell-like tumor cells were demonstrated ultrastructurally. As suggested by other the expression of S-100 protein in investigators,g*“-‘” MPNSTs seems to be related to Schwann cell-like differentiation of the tumor cells. An epithelial marker, EMA, also has been found to be an effective marker of perineurial cells.20~22 It has been detected in perineurial cells of normal peripheral nerves and in some types of BPNSTs, such as localized hypertrophic neuropathy and neuroma. Many ultrastructural studies have shown that perineurial cells are the principal components of neurofibromas’r*’ but, unexpectedly, most of these tumors were devoid of EMA immunoreactivity.*““’ A few EMA-positive MPNSTs have been reported.‘2*‘6*2s One malignant glandular schwannoma showed EMA reactivity along the inner surface of glandular elements,‘* but this epithelial differentiation was not demonstrated in other EMA-positive MPNSTS.‘~,** In the present study the cellular whorls composed of perineurial cells in case no. 1 were weakly reactive. In addition, some primitive cells and Schwann cell-like tumor cells showed EMA reactivity. On the contrary, the cells resembling perineurial cells in case no. 6 were not reactive. Thus, the expression of EMA in cells of MPNSTs varied greatly and was observed not only in well-differentiated perineurial cells but also, occasionally, in Schwann cells and primitive cells. Recent immunohistochemical studies on acoustic schwannomas have demonstrated that Schwann cell-like tumor cells are focally positive for EMA as well as S-100 protein.*’ A similar phenomenon was observed on immunostaining for Leu-7. Anti-Leu-7 monoclonal antibody cross-reacts with myelin-associated glycoprotein and thus has been used as a Schwann cell marker.‘,i4,” In the present study, however, Leu-7 immunoreactivity was demonstrated in primitive cells and the cells resembling perineurial cells. With regard to the cytoskeletons of peripheral nerve sheath tumors, most MPNSTs in the present study were diffusely positive for vimentin, as also found in previous studies.‘“.‘” None showed GFAP reactivity. Schwannomas and neurofibromas often have been found to show GFAP immunoreactivity, but the proportion of MPNSTs showing GFAP reactivity is very low. ‘,“The expression of actin in MPNSTs has been controversial. In one study” actin reactivity was observed in all tumors studied, whereas in another study”’ it was not found in any 869
HUMAN PATHOLOGY
typic expression may suggest a close relationship these cell types.
Volume 23, No. 8 (August
1992)
iJtl22. Theaker JM, Mattel- KC:, Puddle J: Epithelial membrane tigfn expr-ession by the perineurium of peripheral nerve and in pcripherd nel-ve tumours. Histopathology 13: 17 l- 179, 1988 23. Hirose T, Sumitomo M, Kudo E, et al: Malignant peripheral nerve sheath tumor (MPNST) showing perineurial cell differentiation. Am J Surg Pathol 13:613-620, 1989 24. Ghadially FN: Is it a schwannoma or a fibroblastic neoplasm? in Diagnostic Electron Microscopy of TUJJlOUrS (ed 2). London, UK, Butterworth, 1985, pp 206-227 25. Erlandson RA: Peripheral nerve sheath tumors. Ultrastruct Pathol 9:113-122 , 1985. 26. Takahashi K, Yamaguchi H, Ishizeki J, et al: Immunohistochemical and immunoelectromnicroscopic localization of S-l 00 protein in the interdigitating reticulum cells of the human lymph node. Virchows Arch [B] 37:125-135, 1981 27. Hirose T, Sano T, Hizawa K: Ultrastructural localization of S-100 protein in neurofibroma. Acta Neuropathol (Berl) 69:103-l 10, 1986 28. Salisbury JR, Isaacson PG: Synovial sarcoma: An immunohistochemical study. J Path01 147:49-57, 1985 29. Winek RR, Scheithauer BW, Wick MR: Meningioma, meningeal hemangiopericytoma (angioblastic meningioma), peripheral hemangiopericytoma, and acoustic schwannoma. A comparative immunohistochemical study. Am J Surg Path01 13:25 l-261, 1989 30. Miettinen M: Antibody specific to muscle actins in the diagnosis and classification of soft tissue tumors. Am J Pathol 130:205215, 1988 3 1. Skalli 0, Gabbiani G, Babai F, et al: Intermediate filament proteins and actin isoforms as markers for soft tissue tumor differentiation and origin. II. Rhabdomyosarcomas. Am J Path01 130:515531,1988 32. Leader M, Pate1 J, Makin C, et al: An analysis of the sensitivity and specificity of the cytokeratin marker CAM 5.2 for epithelial turnout-s. Results of a study of 203 sarcomas, 50 carcinomas and 28 malignant melanomas. Histopathology 10: 13 15-l 324, 1986 33. Ortonne J-P, Verrando P, Pautrat G, et al: Lamellar cells of sensor-y receptors and perineurial cells of nerve endings of pig skin contain cytokeratins. Virchows Arch [A] 410:547-552, 1987 34. Coggi G, Dell’Orto P, Braidotti P, et al: Coexpression of intermediate filaments in normal and neoplastic human tissues: A reappraisal. Ultrastruct Path01 13:50 l-5 14, 1989 35. Gould VE: The coexpression of distinct classes of intermediate filaments in human neoplasms. Arch Path01 Lab Med 109:984-985, 1985 36. McGuire I,J, Ng JPW, L.ee JCK: &expression of cytokeratin and vimentin. Appl Pathol 7:73-84, 1989 97. Sumitomo M, Hirose T, Kudo E, et al: Epithelial diflerentiation in synovial sarcoma. Correlation with histology and immunophenotypir expression. Acta Pathol Jpn 39:381-387, 1989 38. Daimaru Y, Hashimoto H, Tsuneyoshi M, et al: Epithelial profile of epithelioid sarcoma. An immunohistochemical analysis of eight cases. Cancer 59:134-141, 1987 39. Miettinen M: Immunoreactivity for cytokeratin and epithelial JTleJllbJ2ne antigen in leiomyosarcoma. AI-ch Pathol Lab Med 112: 637-640, 1988 40. Miettinen M, Rapola J: Immunohistochemical spectrum of rhabdomyosarcoma and rhabdomyosarcoma-like tumors. Expression of cytokeratin and the 68-kD neurofilament protein. Am J Surg Pathol 13:120-132, 1989 4 1. Mall R, Lee I, Gould VE, et al: Immunocytochemical analysis of Ewing’s tmiiors. Patterns of expression of intermediate filaments and desmosomal proteins indicate cell type heterogeneity and pluripotential difkreJltiatiOJ1. AJ~ J Pathol 127:288-304, 1987 42. Hirosr T, Kudo E, Hasegawa ‘I‘, et al: Expression of intermediate filaments in malignant fibrous histiocytomas. HUM PA-THOI. 20:871-877, 1989 43. Gray MH, Rosenberg AE, Dickersin GR, et al: Cytokeratin expression in epithelioid vascular neoplasms. HUM PATHOI. 21:2 12217, 1990
among
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870
Study in Relation to
TAKANORI HIROSE, MD, TADASHI HASEGAWA, KUNIHIKO SEKI, MD, TOSHIAKI SANO, MD, AND KAZUO HIZAWA, MD The constituent cells in malignant peripheral nerve sheath tumors were examined by studying the expression of immunohistochemical markers for Schwann cells and perineurial cells in relation to ultrastructural features in 12 malignant peripheral nerve sheath tumors. Ultrastructural studies demonstrated mixed proliferation of Schwann cells, perineurial cells, fibroblastic cells, and primitive cells in many malignant peripheral nerve sheath tumors. Expression of S-100 protein was well correlated with Schwann cell-like differentiation of tumor cells. However, Leu-7 and epithelial membrane antigen, which have been considered to be specific to Schwann cells and perineurial cells, respectively, were common to Schwann cells, perineurial cells, and primitive cells. The common immunophenotypic expression suggests a close relationship among these cell types. The unusual expression of cytokeratin could be explained by the plasticity of intermediate filament expression. HUM PATHOL 23:865-870. Copyright 0 1992 by W.B. Saunders Company
Malignant peripheral nerve sheath tumors (MPNSTs) are poorly defined sarcomas and show great histologic variation.’ Ultrastructural and immunohistochemical studies are necessary for accurate diagnosis of this type of tumor, unless the tumor is associated with neurofihromatosis type 1 or has continuity with a nerve. Previous ultrastructural studies have indicated a Schwann cell nature of the cells in MPNSTs.“.’ These tumors have been found to contain other cell types derived from the peripheral nerve sheath as well, such as heterperineurial cells7-” We also have demonstrated ogeneous cell populations in six MPNSTs by S-l 00 protein-immunostaining and ultrastructural studies.‘” Recently, there have been many immunohistochemical S-100 protein, Leu-7, myelin studies of MPNSTs.“-‘* basic protein, and glial fibrillary acidic protein (GFAP) have been considered to be immunohistochemical markers of Schwann cells in the peripheral nervous system.‘” E P ithelial membrane antigen (EMA) has been demonstrated to be localized in the perineurium of peripheral nerves and has been used as a perineurial cell marker in studies of benign peripheral nerve sheath tuan mors (RPNSTS).~~-~’ We previously have reported MPNST showing diffuse EMA immunoreactivity.” In the present study we examined the irnmunohistochem-
MD, EIJI KUDO, MD,
ical expression of these markers of Schwann cells and perineurial cells as well as some cytoskeletal proteins in 12 tumors, and correlated the results with the ultrastructural features of the tumors.
MATERIALS
AND METHODS
Twelve tumors with characteristic ultrastructural features of MPNSTS’~,‘~ were selected for this study. We have reported the ultrastructural features of seven of these tumors (Table 1; cases no. 2, 3, 6, 7, 9, 10, and 11).‘“,23 Three patients (cases no. 1, 3, and 11) had neurofibromatosis type 1 and in three patients (cases no. 3, 6, and 9) the tumors had continuity with nerves. For immunohistochemical studies, in addition to hematoxylin-eosin staining, 4 pm-thick sections from specimens fixed in 10% formalin and embedded in paraffin were prepared. Immunohistochemical staining was performed using the avidinbiotin-peroxidase complex method with an ABC kit (Vector Laboratories, Burlingame, CA). The primary antibodies used are shown in Table 1. Polyclonal anti-S-l 00 protein antibody (diluted 1:2,000; Takahashi et alz6) and monoclonal anti-leu7 antibody (diluted 1:50; Becton Dickinson, Mountain View, CA) were used as Schwann cell markers, and monoclonal antiEMA antibody (diluted 1:50; DAKOPATTS a/s, Glostrup, Denmark) was used as a perineurial marker. To study the cytoskeletal proteins of MPNSTs we used monoclonal anti-olsmooth muscle actin (diluted 1: 1,000; BioMakor, Rehovot, Israel) and vimentin (diluted 1: 10; DAKOPATIS) antibodies, as well as polyclonal anti-GFAP (diluted 1:500; DAKOPATTS) and neurofilament (200 kd, diluted 1: 100; our laboratory) antibodies. For the study of cytokeratin five different monoclonal antibodies were used: PKKl (44, 46, 52, and 54 kd, diluted 1:lOO; Labsystems, Helsinki, Finland), CAM 5.2 (39 and 43 kd, diluted 1: 1; Becton Dickinson), 35PH 11 (54 kd, diluted 1: 1,000; Enzo, New York, NY), 34PE12 (57 and 66 kd, diluted l:l,OOO; Enzo), and 34PB4 (68 kd, diluted l:l,OOO; Enzo). External lamina was examined with polyclonal anti-type IV collagen antibody (diluted 1:200; Advance, Tokyo, Japan). Immunostaining with anticytokeratin and anti-type IV collagen antibodies required predigestion of the sections with trypsin. For electron microscopic studies specimens of primary, recurrent, or metastatic tumors obtained at surgery were cut into small cubes and fixed in 3% glutaraldehyde in phosphate buffer. The specimens were then washed with buffer solution, postfixed in 1% osmium tetroxide in phosphate buffer, dehydrated, and embedded in Epon 812. For correlation with immunohistochemical findings blocks containing areas that were microscopically similar to immunohistochemical sections were selected. Thin sections from two to nine blocks (mean, 5.8 blocks) of each tumor were stained with uranyl acetate and lead citrate, and examined with an electron microscope. We correlated neoplastic cells of each tumor with the cells
From the First Department of Pathology, University of Tokushima School of Medicine, Tokushima, Japan. Accepted for publication September 30, 1091. Key umrd~: peripheral nerve sheath tumor, malignant, ultrastructure, imnlunohistochemistry. Address correspondence and reprint requests to Takanori Hirose, MD, The First Department of Pathology, University of Tokushima School of Medicine, Kuramoto-cho 3, Tokushima 770, Japan. Copyright C 1992 by W.B. Saunders Company 00468177/92/2308-0005$500/O
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TABLE 1.
Clinical and lmmunohistochemical
Volume 23, No. 8 (August
1992)
Features of Malignant Peripheral Nerve Sheath Tumors Cytokeratin
Case
Age (yr)/ Sex
Location of Tumor
SlOO
Leu-7
EMA
VIM
CAM
PKK
1 2
46/F 75/M
R upper arm R scapular
++ ++
+ ~
+ ~
++ ++
-
_
3
30/F
+
_
~
++
+
+
+
4 5 6 7 8 9
55/M 63/F 37/M 52/M 65/F 20/F
+ ++ _
_
+
+ _ _
++
+ + -
_ _ _ _
_
++ + ++ t+ ++ ++
_
~ + _
_
_
10 11 12
23/F 60/F 58/M
L brachial plexus Prostate L chest wall R chest wall L back R chest wall R brachial plexus I, elbow Ankle L back
_ _ _
+
t
_
t _ _
+
_ _
_ _
5
4
5
11
3
No.
region
Total
_ ++ ~ +
Symbols: ++, dilhtsely positive; +, focally positive; -, negative. Abbreviations: EMA, epithelial membrane antigen: VIM. vimentin; ASMA. a-smooth muscle actin.
constituting normal peripheral nerve sheath and BPNSTs: Schwann cells, perineurial cells, and fibroblasts.“5~27 RESULTS Light Microscopic
34/3B4
GFAP
NF200
ASMA
Type 4 Collagen
_ _
_ _
_ _
_
+ +
+
_
_
_
_
+
_ _
_ _
_ _
_ _
_ _ _ _ _ -
_ _ _ _ _ _
_ _ _ _ _
_ _ _
_ _ _
_ _ _
_ _ _
_ _ -
_ _ _
+ _
1
2
1
0
0
0
0
10
GFAP,
glial fibrillary
35PEll
34PE12
_
acidic
protein;
NF200,
neurofilament
_ t t + + +
+
200KD;
1, center). In case no. 7 spindle-shaped cells were arranged in storiform and whorl patterns.*’ Five tumors (cases no. 8, 9, 10, 11, and 12) consisted entirely of poorly differentiated, short, spindle-shaped cells showing a vague fascicular pattern (Fig 1, right).
Findings
All tumors were principally composed of spindle tumor cells arranged in intersecting fascicles, but there was great histologic variation. Small parts of three tumors (cases no. 1,2, and 3) had neurofibroma-like areas in which spindle cells with wavy nuclei grew irregularly in fibromyxoid stroma (Fig 1, left). In case no. 1 there were many cellular bundles surrounded by perineuriumlike whorls. Nuclear palisading and epithelioid tumor cells were noted in cases no. 4 and 5, respectively (Fig
lmmunohistochemical Findings and Related Ultrastructural Features The results of immunostaining of the 12 MPNSTs are shown in Table 1. S-l 00 protein immunoreactivity was demonstrated in five tumors (cases no. 1, 2, 3, 4, and 5). Many wavy spindle cells in neurofibroma-like areas of cases no. 1, 2, and 3 were stained, whereas poorly differentiated short-spindle cells were not. In neurofibroma-like areas electron microscopy disclosed
FIGURE 1. (Left) A neurofibroma-like area of case no. 3 showing an irregular growth of wavy spindle cells in ftbromyxoid stroma. (Magnification X 174.) (Center) Case 4. An MPNST showing well-developed nuclear palisading. (Magnification x174.) (Right) Case no. 9. Fascicular growth of short, spindle-shaped tumor cells (Magnification x 174.)
MALIGNANT
PERIPHERAL NERVE SHEATH TUMORS
mixed proliferation of Schwann cells, perineurial cells, and fibroblastic cells. Schwann cells, covered by distinctive external laminas (Fig 3a), had broad and occasionally branching cell processes and numerous intermediate filaments. Many S-l 00 protein-positive spindle cells were diffusely distributed in cases no. 4 and 5 (Fig 2a), both of which were predominantly composed of Schwann cell-like cells with numerous cytoplasmic processes entangled and connected with one another (Fig 3b). Leu-7 immunoreactivity was often detected in areas of poorly differentiated, short, spindle cells (cases no. 1, 9, and 10; Fig 2b). Ultrastructural examination of these areas showed that the compact tumor cells had a few short cell processes with no interdigitation and were often connected by primitive junctions. Electron-dense material was seen in the intercellular space (Fig 3~). Because these short, spindle-shaped tumor cells showed no apparent differentiation into Schwann cells or peri-
FIGURE 2. Immunohistostainings of chemical MPNSTs. (a) Case no. 4. Many S-IdO’protein-positive tumor cells. (Magnification x129.)(b) Case 9. Membranous immunoreactivity for Leu-7 on numerous primitive tumor cells. (Magnification X344.) (c) Case no. I. Perineurium-like cellular whorls showing weak reactivity for EMA. (Magnification x172.) (d) Case no. 10. Fine, granular immunoreactions for EMA localized in cell membranes of many primitive tumor cells. (Magnification x258.) (e) Case no. 5. Many scattered tumor cells with cytokeratin (CAM 5.2)-reactive cytoplasm. (Magnitication ~258.) (f) Case no. 3. A few plump tumor cells showing high molecular weight cytokeratin (34pE12) immunoreactivity. (Magnification x172.)
(Hirose et al)
neurial cells, the tumor cells were designated as primitive cells. Some spindle-shaped cells of the cellular fascicles of case no. 6 also showed Leu-7 immunoreactivity; this tumor consisted of perineurial-like cells with long, slender cell processes covered by well-developed external laminas and some pinocytotic vesicles along the cell membrane (Fig 3d). Epithelial membrane antigen-positive tumor cells were present in five tumors. In case no. 1 reaction products were localized in cellular whorls resembling the perineurial sheath found in neurofibroma-like areas (Fig 2c) and these cells showed ultrastructural characteristics of perineurial cells (Fig 3e). Small aggregates of primitive cells (cases no. 9 and 10; Fig 2d) and spindle-shaped cells (case no. 4), which were ultrastructurally similar to Schwann cells (Fig 3b), showed reactivity along their cell membranes, whereas the tumor cells of case no. 7 showed diffuse reactivity. The ultrastructural features of the cells in case no. 7 seemed to
HUMAN PATHOLOGY
Volume 23, No. 8 (August
1992)
FIGURE 3. (a) Case no. 3. Mixed proliferation of a Schwann cell (S) covered by a continuous external lamina and ftbroblastic ceils (F) with well-developed rough endoplasmic reticulum. The arrow indicates long-spacing collagen. (Magnification x6.240.) (b) Case no. 4. Closely packed Schwann cell-like tumor cells with numerous interdigitating cell processes. (Magnification x7.800.) (c) Case no. 9. An ultramicrograph of primitive tumor cells, occasionally connected by primitive junctions (arrowhead). Note electron-dense substances in the intercellular space. (Magnification ~7,800.) (d) Case no. 6. Perineurial-like cells with slender cell processes covered by well-developed external laminas. A few pinocytotic vesicles (arrowheads) are seen along the cell membrane. (Magnification x 13,260.) (e) Case no. I. Perineurial tumor cells characterized by long, slender cell processes, pinocytotic vesicles, and external laminas. (Magnification x13,260.)
lial differentiation was detected in any tumor ultrastructurally. No tumor reacted with anti-cu-smooth muscle actin antibody, which showed intense immunoreaction in vascular smooth muscle cells. Linear, pericellular reactions for type IV collagen were demonstrated in 10 tumors.
be intermediate between those of perineurial and Schwann cells.“’ Vimentin reactivity was observed in all tumors except that in case no. 11, but no reactivity for GFAP or neurofilament 200 kd (both of which were localized in nonneoplastic nerve fibers in the tumors) was detected in any tumor. A few cytokeratin-positive cells were unexpectedly detected in three tumors (cases no. 3, 5, and 6) stained with CAM 5.2. The positive cells, which were spindle-shaped or epithelioid, were scattered among negative cells and were not arranged in any epithelial pattern, such as a nest or tubule (Fig 2e). Other anticytokeratin antibodies (35PH 11, 34PE12, and PKKl) also reacted with a few tumor cells in two, one, and one tumors (Fig 2I), respectively, whereas no immunoreaction with 34PB4 was demonstrated. No definite epithe-
DISCUSSION Because they have no specific microscopic features and show great histologic variation,’ MPNSTs are difficult to differentiate from other soft tissue tumors. As found in the present study, the microscopic features of MPNSTs varied from well-differentiated areas with prominent palisading and neurofibroma-like patterns 868
MALIGNANT
PERIPHERAL NERVE SHEATH TUMORS
(Hirose et al)
tumor. We also did not detect actin in any tumor. Actin is present in almost all mammalian cells, where it exists as one of at least six isoforms that show tissue specificity.3’ Thus, it seems necessary to further investigate actin expression using monoclonal antibodies specific for each isoform. Miettinen” also used monoclonal antibodies specific to muscular actin isoforms. Our findings indicate that MPNSTs do not seem to have muscle-type actins, although they may have other isoforms. Differences in actin isoforms may be of practical use in differentiating MPNSTs from myogenic tumors. Some groups have reported a few MPNSTs showing reactivity for cytokeratin.‘7,28,32 In the present study cytokeratin immunoreactivity was demonstrated in three of 12 tumors studied. These three tumors were diagnosed as MPNSTs from their ultrastructural features and immunoreactivity for S-100 protein and Leu-7. Their unusual expression of cytokeratin might be explained by epithelial differentiation of tumor cells, as suggested by other investigators.32 Malignant peripheral nerve sheath tumors have been shown occasionally to contain epithelial elements with well-develo ed glands and epithelial nests as heterotopic elements P; however, the cytokeratin-positive cells found in the present study were spindle-shaped or polygonal and did not show any epithelial structures microscopically or ultrastructurally. Moreover, none of the three cytokeratin-positive tumors showed reactivity for EMA. Recently, Ortonne et al33 reported cytokeratin immunoreactivity in lamellar cells of sensory receptors and perineurial cells of pig skin. If similar expression occurs in humans, cytokeratin reactivity might be regarded as indicating perineurial cell differentiation of tumor cells. However, the normal perineurium included in the sections examined in the present study did not react with monoclonal anti-cytokeratin antibodies. Another explanation for the presence of cytokeratin, which seems to be the most reasonable, is the plasticity of intermediate filament expression.34s35 The expression of intermediate filaments has been thought to be relatively specific and stable for given cell types, but many cases of unusual or unexpected expression of intermediate filaments in normal, reactive, and neoplastic tissues have been reported. Cytokeratin expression has been observed in many fetal and adult mesenchymal cells, such as endothelial cells and smooth and striated muscle cells,“4s36 as well as in many types of sarcomas, such as synovial sarcoma,37 epithelioid sarcoma,38 leiomyosarcoma,3” rhabdomyosarcoma,40 Ewing’s sarcoma,41 malignant fibrous histiocytoma,42 and epithelioid angiosarcoma.43 In sarcomas the presence of a few cytokeratin-positive tumor cells might be interpreted as a nonspecific shift of intermediate filaments induced by some unknown regulatory mechanisms connected with intermediate filament expression. Ultrastructurally, MPNSTs seem to be composed of Schwann cells, perineurial cells, and primitive cells in varying proportions. The immunohistochemical expression of Leu-7 and EMA, which is considered to be specific to Schwann cells and perineurial cells, respectively, was common to Schwann cells, perineurial cells, and primitive cells. The common immunopheno-
to poorly differentiated areas containing only diffusely distributed primitive cells. These different growth patterns were often intermingled in the same tumor. The ultrastructural features of MPNSTs also vary greatly. Some previous studies have indicated fine structural similarities between the cells of MPNSTs and Schwann cells,‘” whereas other studies have indicated the presence of other cell types as well.7-g We also reported the presence of perineurial cells, fibroblastic cells, and primitive cells (as well as Schwann cells) in MPNSTs.” In the present study we examined the correlation of ultrastructural findings with the expressions of immunohistochemical markers. S-100 protein, which is thought to be restricted to Schwann cells in normal peripheral nerves and neurofibromas,“,” was detected in five tumors in which Schwann cell-like tumor cells were demonstrated ultrastructurally. As suggested by other the expression of S-100 protein in investigators,g*“-‘” MPNSTs seems to be related to Schwann cell-like differentiation of the tumor cells. An epithelial marker, EMA, also has been found to be an effective marker of perineurial cells.20~22 It has been detected in perineurial cells of normal peripheral nerves and in some types of BPNSTs, such as localized hypertrophic neuropathy and neuroma. Many ultrastructural studies have shown that perineurial cells are the principal components of neurofibromas’r*’ but, unexpectedly, most of these tumors were devoid of EMA immunoreactivity.*““’ A few EMA-positive MPNSTs have been reported.‘2*‘6*2s One malignant glandular schwannoma showed EMA reactivity along the inner surface of glandular elements,‘* but this epithelial differentiation was not demonstrated in other EMA-positive MPNSTS.‘~,** In the present study the cellular whorls composed of perineurial cells in case no. 1 were weakly reactive. In addition, some primitive cells and Schwann cell-like tumor cells showed EMA reactivity. On the contrary, the cells resembling perineurial cells in case no. 6 were not reactive. Thus, the expression of EMA in cells of MPNSTs varied greatly and was observed not only in well-differentiated perineurial cells but also, occasionally, in Schwann cells and primitive cells. Recent immunohistochemical studies on acoustic schwannomas have demonstrated that Schwann cell-like tumor cells are focally positive for EMA as well as S-100 protein.*’ A similar phenomenon was observed on immunostaining for Leu-7. Anti-Leu-7 monoclonal antibody cross-reacts with myelin-associated glycoprotein and thus has been used as a Schwann cell marker.‘,i4,” In the present study, however, Leu-7 immunoreactivity was demonstrated in primitive cells and the cells resembling perineurial cells. With regard to the cytoskeletons of peripheral nerve sheath tumors, most MPNSTs in the present study were diffusely positive for vimentin, as also found in previous studies.‘“.‘” None showed GFAP reactivity. Schwannomas and neurofibromas often have been found to show GFAP immunoreactivity, but the proportion of MPNSTs showing GFAP reactivity is very low. ‘,“The expression of actin in MPNSTs has been controversial. In one study” actin reactivity was observed in all tumors studied, whereas in another study”’ it was not found in any 869
HUMAN PATHOLOGY
typic expression may suggest a close relationship these cell types.
Volume 23, No. 8 (August
1992)
iJtl22. Theaker JM, Mattel- KC:, Puddle J: Epithelial membrane tigfn expr-ession by the perineurium of peripheral nerve and in pcripherd nel-ve tumours. Histopathology 13: 17 l- 179, 1988 23. Hirose T, Sumitomo M, Kudo E, et al: Malignant peripheral nerve sheath tumor (MPNST) showing perineurial cell differentiation. Am J Surg Pathol 13:613-620, 1989 24. Ghadially FN: Is it a schwannoma or a fibroblastic neoplasm? in Diagnostic Electron Microscopy of TUJJlOUrS (ed 2). London, UK, Butterworth, 1985, pp 206-227 25. Erlandson RA: Peripheral nerve sheath tumors. Ultrastruct Pathol 9:113-122 , 1985. 26. Takahashi K, Yamaguchi H, Ishizeki J, et al: Immunohistochemical and immunoelectromnicroscopic localization of S-l 00 protein in the interdigitating reticulum cells of the human lymph node. Virchows Arch [B] 37:125-135, 1981 27. Hirose T, Sano T, Hizawa K: Ultrastructural localization of S-100 protein in neurofibroma. Acta Neuropathol (Berl) 69:103-l 10, 1986 28. Salisbury JR, Isaacson PG: Synovial sarcoma: An immunohistochemical study. J Path01 147:49-57, 1985 29. Winek RR, Scheithauer BW, Wick MR: Meningioma, meningeal hemangiopericytoma (angioblastic meningioma), peripheral hemangiopericytoma, and acoustic schwannoma. A comparative immunohistochemical study. Am J Surg Path01 13:25 l-261, 1989 30. Miettinen M: Antibody specific to muscle actins in the diagnosis and classification of soft tissue tumors. Am J Pathol 130:205215, 1988 3 1. Skalli 0, Gabbiani G, Babai F, et al: Intermediate filament proteins and actin isoforms as markers for soft tissue tumor differentiation and origin. II. Rhabdomyosarcomas. Am J Path01 130:515531,1988 32. Leader M, Pate1 J, Makin C, et al: An analysis of the sensitivity and specificity of the cytokeratin marker CAM 5.2 for epithelial turnout-s. Results of a study of 203 sarcomas, 50 carcinomas and 28 malignant melanomas. Histopathology 10: 13 15-l 324, 1986 33. Ortonne J-P, Verrando P, Pautrat G, et al: Lamellar cells of sensor-y receptors and perineurial cells of nerve endings of pig skin contain cytokeratins. Virchows Arch [A] 410:547-552, 1987 34. Coggi G, Dell’Orto P, Braidotti P, et al: Coexpression of intermediate filaments in normal and neoplastic human tissues: A reappraisal. Ultrastruct Path01 13:50 l-5 14, 1989 35. Gould VE: The coexpression of distinct classes of intermediate filaments in human neoplasms. Arch Path01 Lab Med 109:984-985, 1985 36. McGuire I,J, Ng JPW, L.ee JCK: &expression of cytokeratin and vimentin. Appl Pathol 7:73-84, 1989 97. Sumitomo M, Hirose T, Kudo E, et al: Epithelial diflerentiation in synovial sarcoma. Correlation with histology and immunophenotypir expression. Acta Pathol Jpn 39:381-387, 1989 38. Daimaru Y, Hashimoto H, Tsuneyoshi M, et al: Epithelial profile of epithelioid sarcoma. An immunohistochemical analysis of eight cases. Cancer 59:134-141, 1987 39. Miettinen M: Immunoreactivity for cytokeratin and epithelial JTleJllbJ2ne antigen in leiomyosarcoma. AI-ch Pathol Lab Med 112: 637-640, 1988 40. Miettinen M, Rapola J: Immunohistochemical spectrum of rhabdomyosarcoma and rhabdomyosarcoma-like tumors. Expression of cytokeratin and the 68-kD neurofilament protein. Am J Surg Pathol 13:120-132, 1989 4 1. Mall R, Lee I, Gould VE, et al: Immunocytochemical analysis of Ewing’s tmiiors. Patterns of expression of intermediate filaments and desmosomal proteins indicate cell type heterogeneity and pluripotential difkreJltiatiOJ1. AJ~ J Pathol 127:288-304, 1987 42. Hirosr T, Kudo E, Hasegawa ‘I‘, et al: Expression of intermediate filaments in malignant fibrous histiocytomas. HUM PA-THOI. 20:871-877, 1989 43. Gray MH, Rosenberg AE, Dickersin GR, et al: Cytokeratin expression in epithelioid vascular neoplasms. HUM PATHOI. 21:2 12217, 1990
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