Ganglioneuroblastoma, and Neuroblastoma - Europe PMC

Expression

of

Neurofilament Triplet

Proteins in

Human Neural Tumors

An Immunohistochemical Study ofParaganglioma, Ganglioneuroma, -Ganglioneuroblastoma, and Neuroblastoma

MAKIO MUKAI, MD, CHIKAO TORIKATA, HISAMI IRI, MD, YUKIHIKO MORIKAWA, KOICHI SHIMIZU, MD, TADAKAZU SHIMODA, NOBUYUKI NUKINA, MD, YASUO IHARA, and KEIZO KAGEYAMA,

MD, MD, MD, MD, MD

From the Department of Pathology, Keio University School of Medicine, the Division of Surgical Pathology, Clinical Laboratories, Keio University Hospital, the Division of Pathology, Laboratory Medicine, National Children's Hospital, the Department of Pathology, Jikei University School of Medicine, the Department of Neurology, Institute of Brain Research, Faculty of Medicine, University of Tokyo, and the Division of Clinical Physiology II, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan

Intermediate filaments which are specific to neural cells, ie, neurofilaments, consist ofthree subcomponents-68, 150, and 200 kd. Thirty human neural tumors were examined for the presence of these three subcomponents by means of their monospecific antisera. All 8 paragangliomas contained cells that were positive for the 68-kd component, but only 5 of them had cells positive for the 150-kd and 200-kd components. All 4 ganglioneuromas and 11 ganglioneuroblastomas contained cells that reacted with antibodies to all three components. All 7 neuroblastomas had cells reacting with antibody to 68 kd, but only

3 of them had cells that reacted with antibodies to 150 kd and 200 kd. In each case, the number of positive cells depended on the antibody used. The largest number reacting with antibody to 68 kd and the smallest with antibody to 200 kd. Furthermore, it was possible to detect tumor cells in which the 68-kd subcomponent existed by itself, but no tumor cells in which the 150-kd or 200-kd subcomponent existed alone could be detected. These results seem to indicate that antibody to the 68-kd component is sufficiently discriminating to be applied diagnostically. (Am J Pathol 1986, 122:28-35)

INTERMEDIATE filaments are ubiquitous cytoskeletal proteins found in mammalian cells. Five classes of intermediate filaments are recognized on the basis of their distinct biochemical properties, and each class is restricted to certain cell types"2: vimentin in mesenchymal cells, keratin in epithelial cells, glial fibrillary acidic protein in glial cells, desmin in muscle cells, and neurofilament in neural cells. These five types of intermediate filaments are very often used as cell markers in both embryologic and histopathologic studies because of their cell-type specificity.3 Application of antibodies to intermediate filament proteins in the differential diagnosis of tumor has also recently been reported.48 Neurofilaments, present in all neurons of the central and peripheral nervous systems, form part of the neuronal cytoskeleton.9 They are suitable as differentiation

markers because, during embryologic development, they seem to be present exclusively in neural crest and neuroectodermal cells from the earliest appearance of these structures. 10-12 There are some reports on the clinical application of immunohistochemical detection of neurofilaments to human tumors.-3-15 However, emphasis should be placed on the fact that the neurofilaments consist of three subcomponents whose respective molecular weights are approximately 68, 150, and 200 kd.91 6-18 In most of the studies in the field of tumor pathology,"-3-5 unfortunately, monospecific antibodies corresponding to each of the subcomponents have not Accepted for publication July 29, 1985. Address reprint requests to Makio Mukai, MD, Department of Pathology, Keio University School of Medicine, 35Shinanomachi, Shinjuku-ku, Tokyo, Japan 160.

28

Vol. 122

*

NEUROFILAMENT TRIPLET PROTEINS

No. 1

been used. Rather, antibodies to entire neurofilaments have been used. Trojanowski et a19'20 used monospecific antibodies against the triplet proteins in their study, but there is only one case of esthesioneuroblastoma, in which the results obtained for each of the three subcomponents were shown and compared.20 The relationship of these neurofilament triplet proteins to one another, their structural role in neurofilament organization, and their differential functions are only poorly understood. We studied the localization of these three subcomponents in human neural tumors with monospecific antibodies to neurofilament triplet proteins (68 kd, 150 kd, and 200 kd) in order to clarify the differences in distribution between these three subcomponents in tumors and to determine the ways in which these subcomponents can be used in the differential diagnosis of tumors. In addition, this investigation will help in the elucidation of the function and differentiation of these triplet proteins, many aspects of which remain unknown.

29

tion, 1:500). The slides were then exposed to a biotinylated anti-rabbit immunoglobulin antiserum (dilution, 1:500), avidin (dilution, 1:1000), and biotinylated horseradish peroxidase complex.25 The reagents were purchased from Vector Laboratories (Vectastain, Burlingame, Calif). We obtained the peroxidase reaction by incubating the slides in 0.005 % H202 and 0.02 % 3,3'-diaminobenzidine tetrahydrochloride for 10 minutes. Normal rabbit serum and antisera preabsorbed with an excess of the corresponding antigens were used as controls. The method of absorption consisted of incubating a mixture of each antiserum and an excess of the corresponding antigen at room temperature for 1 hour, leaving it at 4 C for a whole day and night, centrifuging it at 10,000 rpm for 20 minutes, and using the supernate for the control study. A similar reaction was induced in 3 alveolar soft part sarcomas (which are difficult to differentiate from paraganglioma), 9 embryonal rhabdomyosarcomas (which are difficult to differentiate from neuroblastoma), and 4 malignant lymphomas.

Materials and Methods A total of 30 patients with neural tumors consisting of 8 paragangliomas, 4 ganglioneuromas, 11 ganglioneuroblastomas, and 7 neuroblastomas were studied. The clinical data for these patients are summarized in Table 1. Surgically removed samples of the tumors were fixed in 10% buffered formalin, embedded in paraffin in the routine manner, and sectioned for immunohistochemical study. Purification of the triplet proteins and immunization have already been described.2" Namely, neurofilaments were purified from rat spinal cords by a modification of the method of Schlaepfer and Freeman."7 After SDS gel electrophoresis, each band of the triplet (68 kd, 150 kd, and 200 kd) was cut out and eluted from the gel with iWo SDS solution. This eluted solution was used to immunize the rabbits. The specificities and characterizations of the antisera were tested by the immunoblotting method. Electrophoretic transfer of proteins on to nitrocellulose sheets was performed according to Towbin et al. Each strip was incubated with appropriately diluted antisera at 4 C overnight and followed by the avidin-biotin complex procedure. Immunohistochemical staining for the triplet proteins was performed as previously described for other antigenic constituents.22 24 That is, the paraffin sections were deparaffinized and treated with 0.1% trypsin in phosphate-buffered saline (pH 7.4) for 30 minutes. Endogenous peroxidase was blocked by incubation of the slides for 10 minutes in 1% NaIO4. After being washed, the slides were incubated with primary antisera (dilu-

.iiix

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200 Kd 1 50 K d

. s;3' .5 -"i' 4 ':'.:

68 Kd

I A BCD Figure 1-The specificites of antisera to 68-kd, 150-kd, and 200-kd subunits were examined by immunoblotting method. Lane A shows SDS gel (10% polyacrylamide) of rat neurofilaments stained with Coomassie brilliant blue. Lanes B, C, and D are the results of transfer of Lane A to the nitrocellulose sheets. Lanes B, C, and D show the results of staining by avidin-biotin complex method using anti-68 kd antiserum, anti-150 kd antiserum, and anti-200 kd antiserum, respectively. It is noted that each antiserum reacts to each antigen (subunit) alone.

30

MUKAI ET AL

AJP * January 1986

Figure 2-Immunohistochemical

staining for the 200-kd component in

sympathetic ganglion. Nerve bundles and ganglion cells are clearly positive, but blood vessels are negative. (Without counterstain,

x 200).

Results

Figure 1 shows the results of the immunoblotting method for checking the specificities and characterization of antisera. Our antisera to 68-kd, 150-kd, and 200kd subunits

reacted to each antigen (subunit)

Normal neural tissues were

nation of the specificity

investigated

of the

antisera.

alone.

for determi-

Antibodies to

all three neurofilament subunits reacted with peripheral nerve axons; the ganglion cells also showed a strong positive reaction (Figure 2). All antibodies reacted to neuronal perikarya and axon. In the immunohistochemical staining of the following human neural tumors, these normal nerve axons and ganglion cells were always used as the positive controls. If nonneoplastic nerve bundles were present in sections from the patient, they were also recognized as positive controls. The controls in which normal rabbit serum or antisera preabsorbed with the corresponding antigens in excess were used showed no reactivity. The results of the study on the localization of the triplet proteins in 30 human neural tumors are shown in Table 1. All 8 paragangliomas showed positive cells when stained with antibody to the 68-kd subunit, but only 5 of them - from the same patients - showed positive cells when stained with antibodies to 150-kd and 200-kd subunits. As shown by staining of serial sections (Figure 3), the antibodies to the 150-kd and 200-kd subunits do not react with all cells that are positively stained with antibody to the 68-kd subunit. All 4 gan-

Table 1-Clinical Data and Localization of Neurofilament Triplet Proteins by Immunohistochemistry Case

Age and

sex

Site

Size (cm)

Follow-up (months)

Neurofilament triplet proteins

Prognosis

Paraganglioma 1 2 3 4 5 6 7 8

Ganglioneuroma 9 10 11

12

Ganglioneuroblastoma 13 14 15

16 17 18 19 20 21 22 23 Neuroblastoma 24 25 26 27 28

29 30

38 M 44 M 46 M 51 F 50 F 42 M 45 F 31 M

Retroperitoneum Retroperitoneum Left neck Retroperitoneum Urinary bladder Left neck Right neck Retroperitoneum

5x3x3.5 6.5x4x 5.5 4x2x2.5 4x3.5x3 3.5x2x2.5 3.5x2.5x2 3x3x2 5.5x4.5x3

23 8 20 4 14 31 19

Alive Alive Alive Alive Alive Alive Alive Alive

6 M 6 F 10 M 7 M

Mediastinum Mediastinum Mediastinum Mediastinum

7x3.5x5 5x5x7 6x4.5x 5.5 7.5x6x6

44 7 16 51

Alive Alive Alive Alive

Retroperitoneum Left adrenal

22

Retroperitoneum Retroperitoneum

8x3x4 15x11 x8 7.5x8x5.5 8x7x4 11 x12x8 12x11 x12 8.5x6x4 8x5x5 5.5x4x4 10.5x8x7 9x9.5x7

Died Died Died Alive Alive Alive Died Alive Died Died Alive

Retroperitoneum Retroperitoneum Retroperitoneum Right adrenal Right adrenal Right adrenal Left adrenal

9.5x3.5x3 4.5 x 5.5 x 3.5 5x3.5x4 8.5x7x6 6x4x4 7.5x6x5 7.5x7x6

4

6 M 3 3 FFM 1 1

F

M F 6 FF 2 F M 2 4

8

M

0

M

0

M

5 F 1 M 2 F 2 F

Left adrenal Left adrenal

Retroperitoneum Retroperitoneum Left adrenal

Retroperitoneum Left adrenal

11

17 9

10 24 18 12 3 13 26 6 8 14 10 13 11

15 9

Alive Alive Died Alive Alive Died Alive

68 kd

150 kd

200 kd

Vol. 122 * No. I


31

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a and b-Serial sections. aFigure 3-Immunohistochemical staining of a paraganglioma with antibodies to the triplet proteins (Case 1). Immunohistochemical staining for 68-kd component without counterstain. b-Immunohistochemical staining for the 150-kd component without counterstain. (x 100) c and d-Serial sections. c-immunohistochemical staining for 68 kd without counterstain. d-lmmunohistochemical staining for 200 kd without counterstain. (x 100) Note that the antibodies to the 150-kd and 200-kd components do not react with all cells that are positively stained with antibody to the 68-kd component.

glioneuromas and 11 ganglioneuroblastomas reacted with antibodies to all three subcomponents of neurofilament (Figures 5 and 6). All 7 neuroblastomas showed positive cells when stained with antibody to 68 kd (Figure 4a), but only 3 of them - from the same patients showed positive cells reacting with antibodies to the 150kd and 200-kd subunits. In other words, positive cells reacting with the antibody to the 68-kd subcomponent were observed in all 30 patients, and those with antibodies to the 150-kd and 200-kd components were observed in 23 patients. Furthermore, in each case, the number of positive cells varied depending on the antibody used. Slides stained with antibody to 68 kd showed the largest number of positive cells; there were fewer cells reacting with antibody to 150 kd, and even fewer cells reacting with antibody to 200 kd, despite the fact that such a difference was not observed in the normal ganglia which were used as the positive controls. A comparison of the serial

sections of the 68-kd, 150-kd and 200-kd subcomponents illustrates this finding (Figures 5 and 6). It was possible to detect tumor cells in which the 68-kd subcomponent existed by itself, but tumor cells in which the 150-kd or 200-kd subcomponents existed alone could not be detected. The 150-kd subcomponent and the 68-kd subcomponent coexisted, and the 200-kd subcomponent coexisted with the 68-kd and 150-kd subcomponents. In ganglioneuromas, ganglioneuroblastomas, and neuroblastomas, the tendency of the cells to show positive staining with antibodies to neurofilament proteins was proportional to the amount of cytoplasm. On the other hand, negative staining was observed in cells with less developed cytoplasm. In small round cells, particularly in ganglioneuroblastoma and neuroblastoma, the 68-kd subcomponent alone was observed (Figure 4). It is of great interest that, in some patients the 68-kd subcomponents alone were detected in the neoplastic nerve

32

MUKAI ET AL

AJP * January 1986

?

4

p

.

f ..

I

1.

4,

*

'p

p

ba-Immunohistochemical staining for the 68-kd component without counterstain. Figure 4-Serial sections of neuroblastoma (Case 24). Immunohistochemical staining for 200 kd without counterstain. (x 100). Note that a large number of tumor cells are positive with antibody to 68 kd, but the nerve bundles located in the central part showed definitely positive staining with the antibodies to 200 kd, while negative staining was observed in all tumor cells.

aFigure 5-Serial sections of glanglioneuroblastoma (Case 17). b-Immunohistochemical staining for the Hematoxylin and eosin. c-Immunohistochemical 150-kd component without counterstain. staining for the 200-kd component without counterstain. Note that 150-kd-positive tumor cells are much more abundant than 200-kd-positive tumor cells. (x 80)

XF,i~*.

33


Vol. 122 * No. 1

!.>

,

5I.;-

aFigure 6-Serial sections of ganglioneuroblastoma (Case 15). b-lmmunohistochemical staining for the 68-kd Hematoxylin and eosin. for c-Immunohistochemical staining the subunit without counterstain. 150-kd subunit without counterstain (arrows indicate positive cells). Note that 68-kd-positive tumor cells are more numerous than 150-kd-positive tumor cells, and that neoplastic nerve bundles are positive for only the 68-kd component. (x 80)

bundles resembling normal nerve bundles in hematoxylin-and-eosin-stained slides (Figure 6). None of the 3 alveolar soft part sarcomas, 9 embryonal rhabdomyosarcomas, and 4 maligant lymphomas that were stained as the controls reacted with the antibodies to neurofilament proteins.

Discussion In this study there was no 150-kd or 200-kd subcomponent in 3 patients with paraganglioma. No characteristics specific to these patients could be found, unlike the other 5 patients.-Although paragangliomas can be classified according to the anatomic distribution and the cellular pattern, no characteristics of the anatomic distribution that were different from those in the other 5 patients could be observed in these 3 patients, as shown in Table 1. As for the cellular pattern, an alveolar tumor pattern was dominant in all 8 patients. Patients in whom other cellular patterns such as an epitheliomatous pattern, a mesenchymomatous pattern,

etc., were dominant were not included in the study. Therefore, the fact that the presence of the 150-kd and 200-kd subcomponents could not be demonstrated in these three patients alone cannot be definitely explained. However, it is believed that the most primitive subcomponent, 68-kd, alone remained and that the 150-kd and 200-kd components disappeared in the process of transformation in these 3 patients. Differences in other aspects between these 3 patients and the other 5 patients could not be detected in this study. The triplet proteins were found in all patients with ganglioneuromas and ganglioneuroblastomas. This phenomenon was due to the constant presence of cells with abundant cytoplasm. There was no 150-kd or 200kd subcomponent in 4 of the 7 patients with neuroblastoma, possibly because there was no cell with relatively abundant cytoplasm, as compared with that in the other 3 patients. However, there were no significant clinical differences between the group with the 150-kd subcomponent and the 200-kd subcomponent and the group without either of these two subcomponents.

34

MUKAI ET AL

The results of the present study revealed that the 68kd subcomponent is clearly the best marker of neural tumors, both from the aspect of the number of patients with positive findings and the number of positive cells. The monospecific antisera used in our present study were those prepared against rat neurofilament immunogens. This fact is important. There have been some reports on the use of monoclonal antibodies against other species of neurofilament immunogens and on the data which are slightly different from ours.26 Positive cells could be detected in all patients examined, and the findings were negative in alveolar soft part sarcoma, malignant lymphoma, and embryonal rhabdomyosarcoma, all of which pose a problem in differential diagnosis. This fact seems to indicate that the antibody to the subcomponent is sufficiently discriminating to be applied diagnostically. Our present data on the 68-kd subcomponent seem to be similar to those obtained with antibodies against the entire neurofilament (not monospecific antibodies for the subcomponents)3-'5 in terms of the number of positive cells and the number of patients with positive findings. In the studies cited, certain antibodies corresponding to the antigenic determinant of the 68-kd subcomponent are considered to have been "accidently" included in the antibodies prepared for the entire neurofilament. Nevertheless, in the future, monospecific antibodies for the 68-kd subcomponent should be prepared and used as a marker in the diagnosis of neural and other tumors. In one report on a case of human esthesioneuroblastoma,20 in which monospecific antibodies were used, the 68-kd subcomponent alone was found, showing agreement with our present data. In addition, our data were compared with other investigators' data concerning the specificity and staining pattern of monospecific antisera. Like in our data, Debus et al2' reported the monoclonal antibodies specifically reacting to each subunit alone. Gown et al,28 on the other hand, reported a monoclonal antibody to the 200-kd subunit, showing cross-reaction with both 68-kd and 160-kd subunits and a monoclonal antibody to the 200-kd subunit showing cross-reaction with the 160-kd subunit alone. Lee et al29 reported four monoclonal antibodies reacting to 200-kd and 150-kd subunits. In addition, Pruss et al30 reported a monoclonal antibody which reacts to not only three subunits of neurofilament but also all other intermediate filaments and suggested that all intermediate filament proteins share a common antigenic determinant. These data reveal that our antisera and the antibodies reported by Debus et al are monospecific to each subunit and are due to the antigenic determinant specific to each subunit.

AJP

*

January 1986

As for staining pattern, there is a report indicating that the 200-kd subunit is stained in the peripheral axon alone.31 According to some reports,32'33 each monoclonal antibody had a unique pattern of staining, reacting only with certain subpopulations of neurons or their processes, as a result of staining of several monoclonal antibodies. On the other hand, there was no difference in each staining pattern among our three antisera in this study. The neuronal perikarya and peripheral axon could be stained in the similar pattern. The monoclonal antibodies reported by Debus et al also showed the same staining pattern. Our antisera are thus considered to be considerably similar to the antibodies reported by Debus et al in terms of specificity and staining pattern. The relationship of the neurofilament triplet proteins to one another, their structural role in neurofilament organization, and their differential functions are only poorly understood. However, some points have been clarified by embryologic investigations34 and in vitro observations35 with rats: a filamentous structure can be formed by the 68-kd subcomponents alone; the 150-kd subcomponents are located so that they cover the whole length of the filamentous structure formed by these 68kd subcomponents; the 200-kd subcomponents recur regularly projecting from the margin of the filament.35 In addition, it is known that the 68-kd and 150-kd subcomponents appear in the early stage of development and that the 200-kd subcomponents appear later and not before the appearance of the other two subcomponents.34 These facts suggest that a very regular genetic program is set for the three kinds of subcomponents. Data from the study of human neural tumors also indicate that the 200-kd subcomponents appear only in the presence of the 68-kd and 150-kd subcomponents, suggesting the maintenance of a regular "set" in the genetic program. However, embryologic studies of the rat brain revealed no nerve cells in which the 68-kd or 150-kd subcomponents exist independently, although the 68-kd and 150-kd subcomponents always coexist.34 In the present study, in contrast, cells in which the 68kd subcomponents existed independently were readily observed, although no cells could be detected in which the 150-kd subcomponents existed independently. Even in mature neoplastic nerve bundles, cells showing the 68-kd subcomponents alone were observed. This fact suggests the possibility that the 68-kd subcomponents are more primitive proteins than either the 200-kd or the 150-kd subcomponents, and that a genetic program for the 150-kd subcomponents is set only in the presence of the 68-kd subcomponents. The expression of the triplet in neural tumors is expected to be applied to investigations of the function and differentiation of neurofilament triplet proteins and embryologic analysis.


Vol. 122 * No. 1

References 1. Franke WW, Schmid E, Osborn M, Weber K: Different intermediate-sized filaments distinguished by immunofluorescence microscopy. Proc Natl Acad Sci USA 1978, 75:5034-5038 2. Lazarides E: Intermediate filaments as mechanical integrators of cellular space. Nature 1980, 283:249-256 3. Schmid E, Tapscott S, Bennett GS, Groop J, Fellini SA, Holtzer H, Franke WW: Different location of different types of intermediate-sized filaments in various tissues of the chick embryo. Differentiation 1979, 15:27-40 4. Miettinen A, Lehto V-P, Badley RA, Virtanen I: Alveolar rhabdomyosdarcoma: Demonstration of the muscle type of intermediate filament protein, desmin, as a diagnostic aid. Am J Pathol 1982, 108:246-251 5. Osborn M, Weber K: Thmor diagnosis by intermediate filament typing: A novel tool for surgical pathology. Lab Invest 1983, 48:372-394 6. Gown AM, Vogel AM: Monoclonal antibodies to human intermediate filament proteins: II. Distribution of filament proteins in normal human tissues. Am J Pathol 1984, 114:309-321 7. Rungger-Brandle E, Gabbiani G: The role of cytoskeletal and cytocontractile elements in pathologic processes. Am J Pathol 1983, 110:361-392 8. Ramaekers FCS, Puts JJG, Moesker 0, Kant A, Huysmans A, Haag D, Jap PHK, Herman CJ, Vooijs GP: Antibodies to intermediate filament proteins in the immunohistochemical identification of human tumours: an overview. Histochem J 1983, 15:691-713 9. Liem RK, Yen SH, Salomon GD, Shelanski ML: Intermediate filaments in nervous tissues. J Cell Biol 1978, 79:637-645 10. Raju T, Bignami A, Dahl D: In vivo and in vitro differentiation of neurons and astrocytes in the rat embryo. Dev Biol 1981, 85:344-357 11. Tapscott SJ, Bennett GS, Holtzer H: Neuronal precursor cells in the chick neural tube express neurofilament proteins. Nature 1981, 292:836-838 12. Tapscott SJ, Bennett GS, Toyama Y, Kleinbart F, Holtzer H: Intermediate filament protein in the developing chick spinal cord. Dev Biol 1981, 86:40-54 13. Lehto V-P, Stenman S, Miettinen M, Dahl D, Virtanen I: Expression of a neural type of intermediate filament as a distinguishing feature between oat cell carcinoma and other lung cancers. Am J Pathol 1983, 110:113-118 14. Lehto V-P, Virtanen I, Miettinen M, Dahl D, Kahri A: Neurofilaments in adrenal and extra-adrenal pheochromocytoma. Arch Pathol Lab Med 1983, 107:492-494 15. Miettinen M, Lehto V-P. Virtanen I: Neuroendocrine carcinoma of the skin (Merkel cell carcinoma): Ultrastructural and immunohistochemical demonstration of neurofilaments. Ultrastruct Pathol 1983, 4:219-225 16. Hoffman PN, Lasek RJ: The slow component of axonal transport: Identification of major structural polypeptides of the axon and their generality among mammalian neurons. J Cell Biol 1975, 66:351-366 17. Schlaepfer WW, Freeman LA: Neurofilament proteins of rat peripheral nerve and spinal cord. J Cell Biol 1978,

78:653-662 18. Thorpe R, Delacourte A, Ayers M, Bullock C, Anderton BH: The polypeptides of isolated brain 10 nm filaments and their association with polymerized tubulin. Biochem J 1979, 181:275-284 19. Trojanowski JQ, Lee VMY: Anti-neurofilament monoclonal antibodies: Reagents for the evaluation of human neoplasms. Acta Neuropathol 1983, 59:155-158 20. Trojanowski JQ, Lee V, Pillsbury N, Lee S: Neuronal origin of human esthesioneuroblastoma demonstrated with

21. 22. 23.

24.

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31.

32.

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35

antineurofilament monoclonal antibodies. N Engl J Med 1982, 307:159-161 Ihara Y, Nukina N, Sugita H, Toyokura Y: Staining of Alzheimer's neurofibrillary tangles with antiserum against 200 k component of neurofilament. Proc Jpn Acad 1981, 57:152-156 Mukai M, Torikata C, Hirose S, Murakami K, Kageyama K: Biochemical and immunohistochemical localization of renin in human pituitary. Lab Invest 1984, 51:425-428 Mukai M, Torikata C, Iri H, Mikata A, Kawai T, Hanaoka H, Yakumaru K, Kageyama K: Histogenesis of clear cell sarcoma of tendons and aponeuroses: An electronmicroscopic, biochemical, enzyme histochemical and immunohistochemical study. Am J Pathol 1984, 114:264-272 Mukai M, Torikata C, Iri H, Hanaoka H, Kawai T, Yakumaru K, Shimoda T, Mikata A, Kageyama K: Cellular differentiation of epithelioid sarcoma: An electronmicroscopic, enzyme-histochemical, and immunohistochemical study. Am J Pathol 1985, 119:44-56 Guesdon JL, Ternynck T, Avrameas S: The use of avidinbiotin interaction in immunoenzymatic techniques. J Histochem Cytochem 1978, 27:1131-1139 Trojanowski JQ, Lee VMY, Schlaepfer WW: An immunohistochemical study of human central and peripheral nervous system tumors, using monoclonal antibodies against neurofilaments and glial filaments. Human Pathol 1984, 15:248-257 Debus E, Weber K, Osborn M: Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides. Differentiation 1983, 25:193-203 Gown AM, Vogel AM: Monoclonal antibodies to human intermediate filament proteins: II. Distribution of filament proteins in normal human tissues. Am J Pathol 1984, 114:309-321 Lee VMY, Page CD, Wu HL, Schlaepfer WW: Monoclonal antibodies to gel-excised glial filament protein and their reactivities with other intermediate filament proteins. J Neurochem 1984, 42:25-32 Pruss RM, Mirsky R, Raff MC, Thorpe R, Dowding AJ, Anderson BH: All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antib6dy. Cell 1981, 27:419-428 Drager UC, Edwards DL, Kleinschmidt J: Neurofilaments contain a-melanocyte-stimulating hormone (a-MSH)-like immunoreactivity. Proc Natl Acad Sci USA 1983, 80:6408-6412 Goldstein ME, Sternberger LA, Sternberger NH: Microheterogeneity ("neurotypy") of neurofilament proteins. Proc Natl Acad Sci USA 1983, 80:3101-3105 Sternberger LA, Sternberger NH: Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. Proc Natl Acad Sci USA 1983, 80:6126-6130 Shaw G, Weber K: Differential expression of neurofilament triplet proteins in brain development. Nature 1982, 298:277-279 Sharp GA, Shaw G, Weber K: Immunoelectron microscopical localization of the three neurofilament triplet proteins along neurofilaments of cultured dorsal root ganglion neurons. Exp Cell Res 1982, 137:403-413

Acknowledgments The authors thank Mr. Shigeo Matsunami for technical assistance. The photographs were prepared by Mr. Koji Takeichi. This manuscript was reviewed by Dr. Mary L. Robbins.