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Intranuclear filaments in a soft tissue sarcoma
INTRANUCLEAR FILAMENTS IN A SOFT TISSUE SARCOMA Frank Gonzalez-Crussi, MD., * Meredith T. Hull, MD., t and David L. Mirkin, MD.*
Abstract Intranuclear filaments aggregating into rodlike structures were found in cells of an undifferentiated soft tissue sarcoma in a child. Similar structures have been uncommonly described in human neoplasms, and uncertainties exist concerning the nature of the inclusion bearing cells in previous reports. The filaments were found to be resistant to mild trypsin digestion. Review of the pertinent literature indicates that these structures may represent the structural manifestation of a highly specialized functional state, rather than a degenerative phenomenon or an artifact. A certain selectivity of occurrence has also been noted. It is therefore plausible to speculate that intranuclear filaments may eventually constitute a morphologic criterion of interest for new tumor classifications.
About one fourth of the malignant solid tumors' of children are sarcomas rising in the soft tissues." A significant proportion of these cannot be confidently classified despite thorough histologic and histochemical study. Their true origin may remain undecided even after the use of specialized techniques, such as tissue culture.! Soule et al. 3 have used the name "sarcoma of undetermined histogenesis" for this group of tumors, and estimated that 27 of 135 childhood cases seen at the Mayo Clinic between 1950 and 1965 deserved this designation. In another large series, nearly 13 per cent of the sarcomas in patients of all ages defied classification." New types of soft t.issue sarcomas have been described in recent years, some with
individualizing features sufficiently consistent to deserve recognition as clinicopathologic entities, and others whose identity is still a matter of debate. In spite of these advances, the relative incidence of sarcoma of undetermined histogenesis in childhood remains important, if difficult to quantitate with accuracy. Cooperative endeavors to unify criteria for diagnosis and treatment, such as those of the Children's Cancer Study Group and the Intergroup Rhabdomyosarcoma Study, have provisionally included this entity with rhabdomyosarcoma of childhood for the practical purpose of evaluating the efficacy of treatment protocols. This should not obscure the fact that some sarcomas exhibit structural features attributed to
"Professor of Pathology, Indiana University School of Medicine. Staff. Department of Pathology. Indiana University Hospital, Indianapolis. Indiana.
t Seuior Resident in Pathology. Indiana University School of Medicine, Indianapolis, Indiana. ~Associate
Professor of Pathology. Indiana University School of Medicine, Indianapolis, Indiana.
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rhabdomyoblastic growths with remarkable consistency, and others fail to show such features in spite of the most exhaustive search. Whereas electron microscopy cannot be expected to clarify the cellular origin in all cases of sarcoma of undertermined histogenesis, it will invariably disclose structural features that otherwise would have been overlooked, and a detailed inventory of which should be compiled for future analysis. We propose to describe here the ultrastructure in a case in which a large number of intranuclear filaments were present and to speculate about the possible meaning of this finding. Case Report
A 14 year old white male presented to a local hospital with a three week history of a left suprascapular mass. The lesion was subjected to biopsy and the biopsy specimen was read as "malignant hemangiopericytoma." The patient subsequently was referred to the James Whitcomb Riley Hospital for Children of the Indiana University Medical Center for further evaluation and definitive therapy. Physical examination at admission was entirely unremarkable aside from a left suprascapular mass, which measured 3.0 by 5.0 by 6.0 em. It was freely movable and minimally tender. Evaluation for metastatic disease, which was negative, included percutaneous liver biopsy, whole lung tomograms, bone scintiscan, liver-spleen scintiscan, and an intravenous pyelogram. Arteriograms revealed that the major vascular supply of the tumor was from the left transverse cervical artery and the left suprascapular artery. The patient underwent complete excision of the mass and experienced an uneventful recovery. Postoperative! y he has been treated with Vincristine, Dactinomycin, and Cytoxan and has remained free of disease for 15 months.
MATERIAL AND METHODS Tissue sections from the excised tumor mass were fixed in buffered formalin, embedded in paraffin, and stained with
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hematoxylin and eosin, Masson's trichrome, Cornori's technique for reticulin, phosphotungstic acid-hematoxylin for demonstration of myofibrils, and periodic acid-Schiff reagent with and without diastase digestion. Small minced tissue fragments, about 1 cu. mm. as used for electron microscopy, were fixed in phosphate buffered 3.5 per cent glutaraldehyde, postfixed in 1 per cent osmium tetroxide, and embedded in epoxy resin. Thick sections (1.0 p,) were cut and stained with 1 per cent toluidine blue and examined by light microscopy. Thin sections were examined either after staining with heavy metals without further manipulation or after the thin sections were floated in a 10 per cent solution of periodic acid, followed by floating in a 0.5 per cent solution of trypsin for 30 minutes, according to the technique described by Monneron and Ber-nhard" for enzymatic digestion of tissues fixed in Epon after double fixation in glutaraldehyde and osmium tetroxide. All sections were stained with lead citrate and uranyl acetate and examined with a Philips 300 electron microscope.
OBSERVATIONS Grossly the tumor was, a uniformly white, soft mass, poorly delimited but distinct from the adjacent muscles. Microscopically the neoplasm was extremely cellular, being made up of abundant, densely packed cells that tended to adopt a fusiform shape. In areas of excessive crowding the cells showed a polyhedral or rounded contour. The cytoplasm was tenuously eosinophilic or amphophilic; the nuclei were remarkably uniform and vesicular and displayed one or two small but conspicuous nucleoli (one often located centrally) set against a light nuclear background. In some areas the tumor showed abundant thin walled blood vessels, with tumor cells often crowding around the vascular walls (Fig. 1). However, no specific perivascular arrangement could be discerned with reticulin stains. Intracytoplasmic myofibrils were not seen. Despite moderate pleomorphism in the most active parts of the lesion, there was
SOFT TISSUE SARCOMA-GoNzALEZ-CRUSSI ET
AL.
Figure 1. Vascular area of the tumor. Note dense cellularity and spindle or polyhedral shape of cells, but no definite perivascular arrangement. (Hematoxylin and eosin stain. X 75.)
no evidence of rhabdomyogenic differentiation. In such areas a high mitosis rate was a feature of the tumor (Fig. 2). By electron microscopy the relatively homogeneous cellular composition of the neoplasm was confirmed. The ovoid shape of the nuclei was revealed to be more irregular than surmised by light microscopy, but no excessive outpouchings or indentations were present. The nuclear envelope was marked by well defined peripheral chromatin, although a fibrous lamina was inconspicuous. The nucleoplasm had abundant but scattered interchromatin granular material, thus correlating with the open faced appearance of the nuclei in histologic preparations (Fig. 3). A striking finding was the presence of elongated rod shaped structures composed of parallel, closely stacked filaments. Most commonly the rods were straight and single. but they also appeared as folded or bent structures with some longitudinal cleaving (Fig'. 4). As measured in electron micrographs. the rods varied between 1.4 and 2.0 micra in length and 0.2 to 0.4
micra in width. Five per cent of the cells in two of five blocks examined contained discernible rods; the remainder of the blocks showed only a rare cell with filaments. In no case was there a visible relationship with the nucleolus. A retrospective review of the Epon embedded t.hick sections did not permit us to identify the rods with absolute certainty, even though. the largest forms fell within the resolving power of the light microscope. The mean diameter of the individual filaments within the roellets was 95 A and the range between 72 and III A (Fig. 5). Cells containing the rods showed no evidence of degenerative phenomena. and commonly were poorly differentiated, as inferred from sparseness of organelles and abundant free ribosomal profiles. Cells with more complex ultrastructure acquired numerous profiles of rough endoplasmic reticulum, Goigi apparatus, andlysosomes or lipid inclusions in their cytoplasm. These cells often contained complex "nuclear bodies" but showed filamentous rod lets only rarely.
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Figure 2. Higher magnification reveals prominent mitotic activity. Nuclei are vesicular. Intranuclear filaments are not identified. (Hematoxylin and eosin stain. x 110.)
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Figure 3. Ultrastructure of the tumor reveals homogeneous cellular composition and undifferentiated appearance of mesenchymal cells. No intranuclear filamen ts are seen in this field. (X 7410.)
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ET
AL.
Figure 4. Intranuclear inclusion composed of parallel filament aggregates. In this cell t.he inclusion is slightly curved and appears longitudinally "split" or frayed. (X 15,580.)
Figure 5. High magnification of filamentous aggregate shows its structural uniformity. Electron density of the individual filaments is comparable to that of neighboring chromatin material. (X 137,000.)
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Figure 6. Following trypsin digestion, much of the cell structure appears to have been replaced by disorganized precipitate of the heavy metal "stains." Intranuclear filaments are still discernible. 67,000.)
ex
Results obtained after trypsin digestion of thick sections showed persistence of the rodlets in some grids after a time sufficient to cause hydrolytic extraction of many cell constituents and some digestion of collagenous fibers (Fig. 6). However, the limited amount of tissue available for electron microscopy did not permit the refining of our estimate of the magnitude of the enzymatic effect by a series of controlled time exposures to digestion activity.
DISCUSSION
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Sarcoma of undetermined histogenesis may be underdiagnosed, partly because surgical pathologists, inured to classifying lesions, tend to equate their inability to do so with an admission of incompetence. In consequence, a tumor difficult to classify may be "forced" into a taxonomic slot either by the expedient of stretching' the criteria for admission, or by that of bestowing excessive importance upon mor-
phologic details that would have been granted a more modest consideration in a less problematic case. Opinions expressed by consultants in our case, and based on light microscopy, reflected this bias in a manner generally applicable to other cases of sarcoma of undetermined histogenesis: Hemangiopericytoma was suggested because of perivascular cell clustering, but it was noted that the reticulin pattern was not in keeping with this diagnosis; synovial sarcoma was suspected because of the location of the mass and the fibrosarcomatous component, but no epithelial component was present; epithelioid sarcoma received consideration, but the histologic character of the lesion was quite unlike that of the prototype cases;" fibrohistiocytic tumors were considered, but the histiocytic nature of the cells could not be confirmed. Consultants at the Armed Forces Institute of Pathology opted for the name "sarcoma, not further classified," a term equivalent to sarcoma of undetermined histogenesis. Accurate definition of histogenesis
SOFT TISSUE SARCOMA- GONZALEZ-CRUSSI
did not seem to be enhanced by ultrastructural study, in terms of the classifications currently in use. Nonetheless, the mesenchymal nature of the cells was confirmed. It may be justified to interpret the range of complexity between primitive polyhedral cells and elongated, organellerich cells of abundant rough endoplasmic reticulum as evidence of fibroblastic differentiation of some cells. It does not follow that there is sufficient reason to designate the tumor as fibrosarcoma, when all the clinical and histologic data are taken into consideration. Thus, with a working diagnosis of sarcoma of undetermined histogenesis, our concern here is to discuss its unusual ultrastructural features. Intranuclear filamentous inclusions have rarely been reported in human neoplasms. Hou-jensen et aLB illustrated their presence in cells tentatively identified as histiocytes, sampled from fibrosing retroperitoneal lesions. Their patient was presumed to have an uncommon form of histiocytosis, with retroperitoneal and mediastinal fibrosis, and a histiocytic lesion ("Gagel's granuloma") of the pituitary gland. More recently intranuclear filamentous bundles were seen in an intrath yroid adenoma with features of hyperparathyroidism.? Lack of adequate preservation in both these studies left many unresolved doubts about the type of cell bearing the intranuclear filament inclusions. We know of no previous documentation of this finding in human soft tissue tumors. Since our material was adequately preserved and showed no necrosis, it is likely that these filaments are neither artifact nor the result of degenerative changes; this interpretation has also been given to structurally identical inclusions in other animal species (to be reviewed). It is also important to note that the samples were obtained before the patient received any form of chemotherapy, since filament stacks in paracrystalline arrays may follow the administration of chemotherapeutic drugs in vitro." A variety of cells are known to possess filaments aggregating in bundles, mostly intracytoplasmic, and presumably made up of a substance related to actin. Criteria for designating filaments as "actinoid" or "actin-like" have included their behavior
ET AL.
following binding with heavy meromyosin and inhibition of this reaction by ATP and pyrophosphate," techniques yielding less than absolute certainty of the chemical nature of the filaments. Published electron micrographs bear at times a striking resemblance to the filaments described in this report. However, circumstantial evidence strongly indicates that intracytoplasmic "actin-like" filaments are structures concerned with the utilization of mechanical forces effecting muscular contraction or cell motility; in specific instances the spatial orientation and rearrangement of the filaments during different phases of the division process indicate that their role concerns such mechanical changes as the furrowing and deformation attending cell cleavage. 10 Filamentous bundles having an intranuclear location are comparatively rare and their significance is less well understood; except in some neurons, it is commonly held that their presence indicates a pathologic condition. In our case the possibility of a cytoplasmic origin of the filaments was considered, because it is known that complex formed particles may on occasion travel between cytoplasm and nucleus. This is a mechanism proposed for filamentous rocUets in neurons. 11 Therefore, filament formation could evince rhabdomyoblastic differentiation of the tumor, of which no hint was present at the light. microscopic level. A proteinaceous composition of the filaments, akin to that of myofibrils, would then be expected. The overwhelming predominance of their intranuclear location and their persistence after trypsin digestion argue against this interpretation, and indirectly favor the intranuclear origin of the filaments under discussion. Several authors l1 - 13 credit Ramon y Cajal for calling attention to elongated intranuclear inclusions in rabbit neurons which he termed "rodlets of Roncoroni," in honor of the Italian investigator who first described these structures. With the advent of electron microscopy their filamentous nature was established.P The occurrence of such inclusions, apparently within normal cells, remains a rather rare event in some cells of the nervous system. Of much greater interest here is the repeated documentation of intranuclear
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filaments in diseases caused by viruses or suspected of having a viral etiology. Filamentous inclusions, indistinguishable from those shown in this report, have been seen in experimental parvovirus hemorrhagic encephalopathy in rats, in cultured HE-p2 cells infected with herpes simplex virus in vitro, in human cases of subacute sclerosing panencephalitis, and in adenovirus type 12-infected human and hamster amnion cells.14 - 17 Typical viral particles mayor may not coexist with filament bundles in the same nucleus in all these infections. Aggregates of filaments have also been well described in cells infected by other types of adenovirus, polyoma virus, herpesvirus horninis-infected mouse cells, swine pox virus, cytomegalovirus, ectromelia virus, and infectious laryngotracheitis virus. 1S- 24 The list is not complete, but it illustrates well the origin of the hypothesis of "guilt by association," namely, that the consistency of this finding justified the conclusion that bundles of intranuclear filaments, whenever present, were a marker of a viral etiology. Despite the finding that some bundles of filaments have been found ultrastructurally to react with antiadenovirus ferritin-labeled anribodies.F the issue is far from resolved. Immunoelectron microscopic procedures have also failed to reveal viral antigens in filaments that structurally were much like paramyxovirus," and a note of caution has been recently introduced by Shaw and Sumi.s" who pointed out the limitations of electron microscopy, as well as the likelihood that unusual intranuclear filaments might represent chromatin threads of a nonviral nature that are apt to be found in diseases for which no evidence exists of a viral etiology. To further compound the problem, the term "filaments" is now applied indiscriminately to such a variety of structures in different structural arrangements that it seems apposite to plead for greater uniformity in nomenclature. In human diseased tissues, intranuclear filaments have been observed most frequently in the brain in cases of multiple sclerosis.f" Both paramyxovirus-like structures and elongated fibrillary bundles or rodlets, identical to those shown here, are present in this disease, singly or in combination within a given cell nucleus.
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The recent statement that intranuclear filaments have a definite selectivity of occurrence in perivascular mononuclear cells in this disease is specially relevant to our discussion." Preferential localization could indicate a greater propensity of cells in a perivascular location to react to abnormal stimuli with formation of intranuclear filamentous material. Alternatively, it could mean that local conditions exist that make perivascular cells a favored target of still undefined noxious stimuli, the latter being otherwise capable of inducing intranuclear filament formation in various types of cells. In either case selectivity of distribution becomes an important issue, for even if the interpretation that regards intranuclear filamentous bundles as "viral footprints" is eventually discarded as too gross a generalization.l? their possible role as a histogenetic marker, or an identifying morphologic trait, would still be worthy of consideration. We do not mean to imply that described rodlets will be a useful and reliable histogenetic marker on which to subordinate the classification of tumors now regarded as sarcoma of uncletermined histogenesis. The known presence of identical inclusions in a wide variety of cells would contradict this reasoning. However, tumor classifications are currently based on morphologic features that in many cases ascribe, by inference, a given histogenesis to neoplastic cells. This inference presupposes that morphology is determined by, or at least the necessary accompaniment of, a functional and metabolic specialization. Intranuclear filaments appearing as "rodlets of Roncoroni" may be assumed to be the ultrastructural correlate of disturbed nuclear metabolism, regardless of whether induced by a viral or a nonviral agency. As such, they might express an abnormality restricted to cells already specialized in a functional sense. For instance, in the endocrine pancreas it is remarkable that the rods should occur only in beta cells of strains of obese diabetic rats, but not in other epithelial islet cells of the same embryonic derivation. 29 Thus, it seems appropriate to speculate that a better definition of the now heterogeneous grou p of sarcomas of undetermined histogenesis could benefit from careful recording of ultrastructural de-
SOFT TISSUE SARCOMA-GONZALEZ-CRUSSI ET AL.
tails. In particular, the work reviewed here suggests that it might be fruitful to scrutinize tumors with a tendency to perivascular cell grouping for the presence of intranuclear filaments. Today "hemangiopericytoma" is more often overdiagnosed than reasonably validated.P'' and reviewers of the subject have concluded that ultrastructural diagnosis of these tumors was unsatisfactory or, at least, equivocal. 31 We believe that patient pursuit of ultrastructural studies should be predicated on the grounds that they provide information of a different order than that on which current classifications were built, and that gradual accretion of this information should provide the basis for new and improved systematizations in the future.
REFERENCES
J. L., Jr., and Miller, R. W.: Incidence of malignant tumors in U.S. children.]. Pediatr. Surg., 86:254, 1975. Margin, R. G., Butler.}. J., and Albores-Saavedra, .J.: Soft Tissue Tumors: Surgical Treatment and Results in Tumors of Bone and Soft Tissue. Chicago, Veal' Book Medical Publishers, In~, 1965,pp.333-347. Soule, E. H., Majour, G. H., Mills, S. D., and Lynn, V. B.: Soft tissue sarcomas of infants • and children: a clinicopathologic study of 135 cases. Mayo Clin. Proc., 43 :313, 1968. Monneron, A., and Bernhard, W.: Action de certaines enzymes sur des tissus inclus en Epon.]. Microsc., 5 :697, 1966. Enzinger, F. M.: Epithelioid sarcoma. Cancer, 26: I 020, 1970. Hou-jensen, K., Rawlison, D. G., and Hendrickson, M.: Proliferating histiocytic lesion (histiocytosis-X?). Cancer, 32:809, 1973. Sherwin, R. P., Kaufman, C., Denner, G. R., and Monroe, S. A.: Intranuclear rodlets in an intrathyroid tumor associated with hyperthyroidism. Cancer, 39: 178, 1977. Krishan, A., and Hsu, D.: Observations on the association of helical polyribosomes and filaments with Vincristine-induced crystals in Earle's L-cell fibroblasts j. Cell Bio!., 43:553, 1969. Ishikawa, H., Bischoff, R., and Holtzer, H.: Formation of arrowhead complexes with heavy meromyosin in a variety of cell types. j. Cell Bio!.,43:312,1969. Forer, A., and Behnke, 0.: An actin-like component in sperrnatocytes of a crane fly (Nephrotorna suturalis Loew). II. The cell cortex. Chromosorna (Ber!.), 39: 175, J 972. Masurovsky, E. B., Benitez, H. H., Kim, S. V., and Murray, M. R.: Origin, development and nature of intranuclear rodlets anel associated
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4. 5. 6. 7.
8.
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bodies in chicken sympathetic neurons. j. Cell Bio!.,44:172,1970. 12. Popoff, N., and Stewart, S.: The fine structure of nuclear inclusions in the brain of experimental golden hamsters. ]. Ultrastruct, Res., 23:347,1968. 13. Siegesmund, K. A., Dutta, C. R., and Fox, C. A.: The ultrastructure of the nuclear rodlet in certain nerve cells. ]. Anat. (Lond.), 98:93, 1964. 14. Baringer, j. R., and Nathanson, N.: Parvovirus hemorrhagic encephalopathy of rats. Electron microscopic observations of the vascular lesions. Lab. Invest., 27:514,1972. 15. Schwartz, ]., and Roizman, B.: Similarities and differences in the development of laboratory strains and freshly isolated strains of herpes simplex virus in HEp-2 cells: electron microscopy.]. Vlrol., 4:879,1969. 16. Oyanagi, S., Rorke, L. B., Katz, M.. and Koprowski, H.: Histopathology and electron microscopy of three cases of subacute sclerosing panencephalitis (SSPE). Acta NeuropathoJ. (Ber!.), 18:58, 1971. 17. Kalnins, U. I., Strich, H. F., Gregory, C., and Gohn, D. S.: Localization of tumor antigens in adenovirus-12-induced tumor cells, and in adenovirus-Iz-infected human and hamster cells by ferritin-labeled antibodies. Cancer Res.,27:1874,1967. 18. Chandler, R. 1..: Demonstration of a porcine adenovirus by electron microscopy. Virology, 25:143,1965. 19. Dourmashkin, R. R.: Electron microscopy of polyoma virus: a review. In Dalton, A. j., and Haguenau, F. (Editors): Tumors Induced by Viruses. New York, Academic Press, Inc., 1962, pp. 151-182. 20. Murphy, F. A., Harrison, A. K., and Whitfield, S. G.: Intranuclear formation of filaments in herpesvirus hominis infection of mice. Arch. Ges. Virusforsch.,21:463, 1967. 21. Reczko, E.: Electronenmikroskopische Vntersuschung del' mit originaren Schweinepoken infizierten Bauchhaut des Ferkels. Arch. Ges. Virusforsch., 9:193, 1959. 22. Ruebner, B. B., Hirano, T., Slusser, R., Osborn, j., and Medearis, D. N.: Cytomegalovirus infection. Viral ultrastructure with particular reference to the relationshi p of liposomes to cytoplasmic inclusions. Am.]. Pathol., 48:970, 1966. 23. Siegel, B. V.: Filamentous structures in ectromelia virus infected cells. Nature, 186:820, 1960. 24. Watrach, A. M.: Intranuclear filaments associated with infectious laryngotracheitis virus. Virology, 18:324,1962. 25. Duboid-Dalcq, M., Schumacher, G., and Sever, J. L.: Acute multiple sclerosis: electron microscopic evidence for and against a viral agent in the plaques. Lancet,2:1408, 1973. 26. Shaw, C. M., and Sumi, S. !vI.: Non-viral intranuclear filamentous inclusions. Arch. Neurol., 32:428, 1975. 27. Tanaka, R, Iwasaki, Y., and Koprowski, H.: Unusual intranuclear filaments in multiple sclerosis brain. Lancet, 1: 1236, 1974.
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I-IUMAN PATHOLOGY - VOLUME 9, NUMBER 2 28. Tanaka, R., Santoli, A., and Koprowski, H.: Unusual intranuclear filaments in the circulating lymphocytes of patients with multiple sclerosis and optic neuritis. Am. J. Pathol., 83: 245,1976. 29. Boquist, 1.: Intranuclear rods in pancreatic islet beta cells.]. Cell. BioI., 43:377, 1969. 30. Silverberg, S. G., Wilson, M. A., and Buard.]. A.: Hemangiopericytoma of the uterus. An ultra-
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structural study. Am . .J. Obstet. Gynecol., 110: 397, 1974. 31. Morales, A. R., Fine, G., Pardo, V., and Horn, R. C.: The ultrastructure of smooth muscle tumors with a consideration of the possible relationship of glomangiomas, hemangiopericytornas, and cardiac myxomas. Pathol. Ann., 10:65, 1975.
Department of Pathology Indiana University School of Medicine 1100 West Michigan Street Indianapolis, Indiana 46202 (Dr. Crussi)
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Abstract Intranuclear filaments aggregating into rodlike structures were found in cells of an undifferentiated soft tissue sarcoma in a child. Similar structures have been uncommonly described in human neoplasms, and uncertainties exist concerning the nature of the inclusion bearing cells in previous reports. The filaments were found to be resistant to mild trypsin digestion. Review of the pertinent literature indicates that these structures may represent the structural manifestation of a highly specialized functional state, rather than a degenerative phenomenon or an artifact. A certain selectivity of occurrence has also been noted. It is therefore plausible to speculate that intranuclear filaments may eventually constitute a morphologic criterion of interest for new tumor classifications.
About one fourth of the malignant solid tumors' of children are sarcomas rising in the soft tissues." A significant proportion of these cannot be confidently classified despite thorough histologic and histochemical study. Their true origin may remain undecided even after the use of specialized techniques, such as tissue culture.! Soule et al. 3 have used the name "sarcoma of undetermined histogenesis" for this group of tumors, and estimated that 27 of 135 childhood cases seen at the Mayo Clinic between 1950 and 1965 deserved this designation. In another large series, nearly 13 per cent of the sarcomas in patients of all ages defied classification." New types of soft t.issue sarcomas have been described in recent years, some with
individualizing features sufficiently consistent to deserve recognition as clinicopathologic entities, and others whose identity is still a matter of debate. In spite of these advances, the relative incidence of sarcoma of undetermined histogenesis in childhood remains important, if difficult to quantitate with accuracy. Cooperative endeavors to unify criteria for diagnosis and treatment, such as those of the Children's Cancer Study Group and the Intergroup Rhabdomyosarcoma Study, have provisionally included this entity with rhabdomyosarcoma of childhood for the practical purpose of evaluating the efficacy of treatment protocols. This should not obscure the fact that some sarcomas exhibit structural features attributed to
"Professor of Pathology, Indiana University School of Medicine. Staff. Department of Pathology. Indiana University Hospital, Indianapolis. Indiana.
t Seuior Resident in Pathology. Indiana University School of Medicine, Indianapolis, Indiana. ~Associate
Professor of Pathology. Indiana University School of Medicine, Indianapolis, Indiana.
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rhabdomyoblastic growths with remarkable consistency, and others fail to show such features in spite of the most exhaustive search. Whereas electron microscopy cannot be expected to clarify the cellular origin in all cases of sarcoma of undertermined histogenesis, it will invariably disclose structural features that otherwise would have been overlooked, and a detailed inventory of which should be compiled for future analysis. We propose to describe here the ultrastructure in a case in which a large number of intranuclear filaments were present and to speculate about the possible meaning of this finding. Case Report
A 14 year old white male presented to a local hospital with a three week history of a left suprascapular mass. The lesion was subjected to biopsy and the biopsy specimen was read as "malignant hemangiopericytoma." The patient subsequently was referred to the James Whitcomb Riley Hospital for Children of the Indiana University Medical Center for further evaluation and definitive therapy. Physical examination at admission was entirely unremarkable aside from a left suprascapular mass, which measured 3.0 by 5.0 by 6.0 em. It was freely movable and minimally tender. Evaluation for metastatic disease, which was negative, included percutaneous liver biopsy, whole lung tomograms, bone scintiscan, liver-spleen scintiscan, and an intravenous pyelogram. Arteriograms revealed that the major vascular supply of the tumor was from the left transverse cervical artery and the left suprascapular artery. The patient underwent complete excision of the mass and experienced an uneventful recovery. Postoperative! y he has been treated with Vincristine, Dactinomycin, and Cytoxan and has remained free of disease for 15 months.
MATERIAL AND METHODS Tissue sections from the excised tumor mass were fixed in buffered formalin, embedded in paraffin, and stained with
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hematoxylin and eosin, Masson's trichrome, Cornori's technique for reticulin, phosphotungstic acid-hematoxylin for demonstration of myofibrils, and periodic acid-Schiff reagent with and without diastase digestion. Small minced tissue fragments, about 1 cu. mm. as used for electron microscopy, were fixed in phosphate buffered 3.5 per cent glutaraldehyde, postfixed in 1 per cent osmium tetroxide, and embedded in epoxy resin. Thick sections (1.0 p,) were cut and stained with 1 per cent toluidine blue and examined by light microscopy. Thin sections were examined either after staining with heavy metals without further manipulation or after the thin sections were floated in a 10 per cent solution of periodic acid, followed by floating in a 0.5 per cent solution of trypsin for 30 minutes, according to the technique described by Monneron and Ber-nhard" for enzymatic digestion of tissues fixed in Epon after double fixation in glutaraldehyde and osmium tetroxide. All sections were stained with lead citrate and uranyl acetate and examined with a Philips 300 electron microscope.
OBSERVATIONS Grossly the tumor was, a uniformly white, soft mass, poorly delimited but distinct from the adjacent muscles. Microscopically the neoplasm was extremely cellular, being made up of abundant, densely packed cells that tended to adopt a fusiform shape. In areas of excessive crowding the cells showed a polyhedral or rounded contour. The cytoplasm was tenuously eosinophilic or amphophilic; the nuclei were remarkably uniform and vesicular and displayed one or two small but conspicuous nucleoli (one often located centrally) set against a light nuclear background. In some areas the tumor showed abundant thin walled blood vessels, with tumor cells often crowding around the vascular walls (Fig. 1). However, no specific perivascular arrangement could be discerned with reticulin stains. Intracytoplasmic myofibrils were not seen. Despite moderate pleomorphism in the most active parts of the lesion, there was
SOFT TISSUE SARCOMA-GoNzALEZ-CRUSSI ET
AL.
Figure 1. Vascular area of the tumor. Note dense cellularity and spindle or polyhedral shape of cells, but no definite perivascular arrangement. (Hematoxylin and eosin stain. X 75.)
no evidence of rhabdomyogenic differentiation. In such areas a high mitosis rate was a feature of the tumor (Fig. 2). By electron microscopy the relatively homogeneous cellular composition of the neoplasm was confirmed. The ovoid shape of the nuclei was revealed to be more irregular than surmised by light microscopy, but no excessive outpouchings or indentations were present. The nuclear envelope was marked by well defined peripheral chromatin, although a fibrous lamina was inconspicuous. The nucleoplasm had abundant but scattered interchromatin granular material, thus correlating with the open faced appearance of the nuclei in histologic preparations (Fig. 3). A striking finding was the presence of elongated rod shaped structures composed of parallel, closely stacked filaments. Most commonly the rods were straight and single. but they also appeared as folded or bent structures with some longitudinal cleaving (Fig'. 4). As measured in electron micrographs. the rods varied between 1.4 and 2.0 micra in length and 0.2 to 0.4
micra in width. Five per cent of the cells in two of five blocks examined contained discernible rods; the remainder of the blocks showed only a rare cell with filaments. In no case was there a visible relationship with the nucleolus. A retrospective review of the Epon embedded t.hick sections did not permit us to identify the rods with absolute certainty, even though. the largest forms fell within the resolving power of the light microscope. The mean diameter of the individual filaments within the roellets was 95 A and the range between 72 and III A (Fig. 5). Cells containing the rods showed no evidence of degenerative phenomena. and commonly were poorly differentiated, as inferred from sparseness of organelles and abundant free ribosomal profiles. Cells with more complex ultrastructure acquired numerous profiles of rough endoplasmic reticulum, Goigi apparatus, andlysosomes or lipid inclusions in their cytoplasm. These cells often contained complex "nuclear bodies" but showed filamentous rod lets only rarely.
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Figure 2. Higher magnification reveals prominent mitotic activity. Nuclei are vesicular. Intranuclear filaments are not identified. (Hematoxylin and eosin stain. x 110.)
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Figure 3. Ultrastructure of the tumor reveals homogeneous cellular composition and undifferentiated appearance of mesenchymal cells. No intranuclear filamen ts are seen in this field. (X 7410.)
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ET
AL.
Figure 4. Intranuclear inclusion composed of parallel filament aggregates. In this cell t.he inclusion is slightly curved and appears longitudinally "split" or frayed. (X 15,580.)
Figure 5. High magnification of filamentous aggregate shows its structural uniformity. Electron density of the individual filaments is comparable to that of neighboring chromatin material. (X 137,000.)
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Figure 6. Following trypsin digestion, much of the cell structure appears to have been replaced by disorganized precipitate of the heavy metal "stains." Intranuclear filaments are still discernible. 67,000.)
ex
Results obtained after trypsin digestion of thick sections showed persistence of the rodlets in some grids after a time sufficient to cause hydrolytic extraction of many cell constituents and some digestion of collagenous fibers (Fig. 6). However, the limited amount of tissue available for electron microscopy did not permit the refining of our estimate of the magnitude of the enzymatic effect by a series of controlled time exposures to digestion activity.
DISCUSSION
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Sarcoma of undetermined histogenesis may be underdiagnosed, partly because surgical pathologists, inured to classifying lesions, tend to equate their inability to do so with an admission of incompetence. In consequence, a tumor difficult to classify may be "forced" into a taxonomic slot either by the expedient of stretching' the criteria for admission, or by that of bestowing excessive importance upon mor-
phologic details that would have been granted a more modest consideration in a less problematic case. Opinions expressed by consultants in our case, and based on light microscopy, reflected this bias in a manner generally applicable to other cases of sarcoma of undetermined histogenesis: Hemangiopericytoma was suggested because of perivascular cell clustering, but it was noted that the reticulin pattern was not in keeping with this diagnosis; synovial sarcoma was suspected because of the location of the mass and the fibrosarcomatous component, but no epithelial component was present; epithelioid sarcoma received consideration, but the histologic character of the lesion was quite unlike that of the prototype cases;" fibrohistiocytic tumors were considered, but the histiocytic nature of the cells could not be confirmed. Consultants at the Armed Forces Institute of Pathology opted for the name "sarcoma, not further classified," a term equivalent to sarcoma of undetermined histogenesis. Accurate definition of histogenesis
SOFT TISSUE SARCOMA- GONZALEZ-CRUSSI
did not seem to be enhanced by ultrastructural study, in terms of the classifications currently in use. Nonetheless, the mesenchymal nature of the cells was confirmed. It may be justified to interpret the range of complexity between primitive polyhedral cells and elongated, organellerich cells of abundant rough endoplasmic reticulum as evidence of fibroblastic differentiation of some cells. It does not follow that there is sufficient reason to designate the tumor as fibrosarcoma, when all the clinical and histologic data are taken into consideration. Thus, with a working diagnosis of sarcoma of undetermined histogenesis, our concern here is to discuss its unusual ultrastructural features. Intranuclear filamentous inclusions have rarely been reported in human neoplasms. Hou-jensen et aLB illustrated their presence in cells tentatively identified as histiocytes, sampled from fibrosing retroperitoneal lesions. Their patient was presumed to have an uncommon form of histiocytosis, with retroperitoneal and mediastinal fibrosis, and a histiocytic lesion ("Gagel's granuloma") of the pituitary gland. More recently intranuclear filamentous bundles were seen in an intrath yroid adenoma with features of hyperparathyroidism.? Lack of adequate preservation in both these studies left many unresolved doubts about the type of cell bearing the intranuclear filament inclusions. We know of no previous documentation of this finding in human soft tissue tumors. Since our material was adequately preserved and showed no necrosis, it is likely that these filaments are neither artifact nor the result of degenerative changes; this interpretation has also been given to structurally identical inclusions in other animal species (to be reviewed). It is also important to note that the samples were obtained before the patient received any form of chemotherapy, since filament stacks in paracrystalline arrays may follow the administration of chemotherapeutic drugs in vitro." A variety of cells are known to possess filaments aggregating in bundles, mostly intracytoplasmic, and presumably made up of a substance related to actin. Criteria for designating filaments as "actinoid" or "actin-like" have included their behavior
ET AL.
following binding with heavy meromyosin and inhibition of this reaction by ATP and pyrophosphate," techniques yielding less than absolute certainty of the chemical nature of the filaments. Published electron micrographs bear at times a striking resemblance to the filaments described in this report. However, circumstantial evidence strongly indicates that intracytoplasmic "actin-like" filaments are structures concerned with the utilization of mechanical forces effecting muscular contraction or cell motility; in specific instances the spatial orientation and rearrangement of the filaments during different phases of the division process indicate that their role concerns such mechanical changes as the furrowing and deformation attending cell cleavage. 10 Filamentous bundles having an intranuclear location are comparatively rare and their significance is less well understood; except in some neurons, it is commonly held that their presence indicates a pathologic condition. In our case the possibility of a cytoplasmic origin of the filaments was considered, because it is known that complex formed particles may on occasion travel between cytoplasm and nucleus. This is a mechanism proposed for filamentous rocUets in neurons. 11 Therefore, filament formation could evince rhabdomyoblastic differentiation of the tumor, of which no hint was present at the light. microscopic level. A proteinaceous composition of the filaments, akin to that of myofibrils, would then be expected. The overwhelming predominance of their intranuclear location and their persistence after trypsin digestion argue against this interpretation, and indirectly favor the intranuclear origin of the filaments under discussion. Several authors l1 - 13 credit Ramon y Cajal for calling attention to elongated intranuclear inclusions in rabbit neurons which he termed "rodlets of Roncoroni," in honor of the Italian investigator who first described these structures. With the advent of electron microscopy their filamentous nature was established.P The occurrence of such inclusions, apparently within normal cells, remains a rather rare event in some cells of the nervous system. Of much greater interest here is the repeated documentation of intranuclear
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filaments in diseases caused by viruses or suspected of having a viral etiology. Filamentous inclusions, indistinguishable from those shown in this report, have been seen in experimental parvovirus hemorrhagic encephalopathy in rats, in cultured HE-p2 cells infected with herpes simplex virus in vitro, in human cases of subacute sclerosing panencephalitis, and in adenovirus type 12-infected human and hamster amnion cells.14 - 17 Typical viral particles mayor may not coexist with filament bundles in the same nucleus in all these infections. Aggregates of filaments have also been well described in cells infected by other types of adenovirus, polyoma virus, herpesvirus horninis-infected mouse cells, swine pox virus, cytomegalovirus, ectromelia virus, and infectious laryngotracheitis virus. 1S- 24 The list is not complete, but it illustrates well the origin of the hypothesis of "guilt by association," namely, that the consistency of this finding justified the conclusion that bundles of intranuclear filaments, whenever present, were a marker of a viral etiology. Despite the finding that some bundles of filaments have been found ultrastructurally to react with antiadenovirus ferritin-labeled anribodies.F the issue is far from resolved. Immunoelectron microscopic procedures have also failed to reveal viral antigens in filaments that structurally were much like paramyxovirus," and a note of caution has been recently introduced by Shaw and Sumi.s" who pointed out the limitations of electron microscopy, as well as the likelihood that unusual intranuclear filaments might represent chromatin threads of a nonviral nature that are apt to be found in diseases for which no evidence exists of a viral etiology. To further compound the problem, the term "filaments" is now applied indiscriminately to such a variety of structures in different structural arrangements that it seems apposite to plead for greater uniformity in nomenclature. In human diseased tissues, intranuclear filaments have been observed most frequently in the brain in cases of multiple sclerosis.f" Both paramyxovirus-like structures and elongated fibrillary bundles or rodlets, identical to those shown here, are present in this disease, singly or in combination within a given cell nucleus.
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The recent statement that intranuclear filaments have a definite selectivity of occurrence in perivascular mononuclear cells in this disease is specially relevant to our discussion." Preferential localization could indicate a greater propensity of cells in a perivascular location to react to abnormal stimuli with formation of intranuclear filamentous material. Alternatively, it could mean that local conditions exist that make perivascular cells a favored target of still undefined noxious stimuli, the latter being otherwise capable of inducing intranuclear filament formation in various types of cells. In either case selectivity of distribution becomes an important issue, for even if the interpretation that regards intranuclear filamentous bundles as "viral footprints" is eventually discarded as too gross a generalization.l? their possible role as a histogenetic marker, or an identifying morphologic trait, would still be worthy of consideration. We do not mean to imply that described rodlets will be a useful and reliable histogenetic marker on which to subordinate the classification of tumors now regarded as sarcoma of uncletermined histogenesis. The known presence of identical inclusions in a wide variety of cells would contradict this reasoning. However, tumor classifications are currently based on morphologic features that in many cases ascribe, by inference, a given histogenesis to neoplastic cells. This inference presupposes that morphology is determined by, or at least the necessary accompaniment of, a functional and metabolic specialization. Intranuclear filaments appearing as "rodlets of Roncoroni" may be assumed to be the ultrastructural correlate of disturbed nuclear metabolism, regardless of whether induced by a viral or a nonviral agency. As such, they might express an abnormality restricted to cells already specialized in a functional sense. For instance, in the endocrine pancreas it is remarkable that the rods should occur only in beta cells of strains of obese diabetic rats, but not in other epithelial islet cells of the same embryonic derivation. 29 Thus, it seems appropriate to speculate that a better definition of the now heterogeneous grou p of sarcomas of undetermined histogenesis could benefit from careful recording of ultrastructural de-
SOFT TISSUE SARCOMA-GONZALEZ-CRUSSI ET AL.
tails. In particular, the work reviewed here suggests that it might be fruitful to scrutinize tumors with a tendency to perivascular cell grouping for the presence of intranuclear filaments. Today "hemangiopericytoma" is more often overdiagnosed than reasonably validated.P'' and reviewers of the subject have concluded that ultrastructural diagnosis of these tumors was unsatisfactory or, at least, equivocal. 31 We believe that patient pursuit of ultrastructural studies should be predicated on the grounds that they provide information of a different order than that on which current classifications were built, and that gradual accretion of this information should provide the basis for new and improved systematizations in the future.
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Department of Pathology Indiana University School of Medicine 1100 West Michigan Street Indianapolis, Indiana 46202 (Dr. Crussi)
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