Ultrastructural Pathology
ISSN: 0191-3123 (Print) 1521-0758 (Online) Journal homepage: http://www.tandfonline.com/loi/iusp20
Osteogenic Sarcoma with Epithelial Differentiation Irving Dardick, Julie E. Schatz & Terence J. Colgan To cite this article: Irving Dardick, Julie E. Schatz & Terence J. Colgan (1992) Osteogenic Sarcoma with Epithelial Differentiation, Ultrastructural Pathology, 16:4, 463-474, DOI: 10.3109/01913129209057831 To link to this article: http://dx.doi.org/10.3109/01913129209057831
Published online: 10 Jul 2009.
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Date: 15 March 2016, At: 12:52
Intriguing Case Osteogenic Sarcoma with Epithelial Differentiation _______
~
_
_
.
_
Irving Dardick, MD, FRCPC Department of Pathology, University of lbronto, Banting Institute, 100 College Street, Ibronto, Ontario M5G lL5, Canada
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
Julie E. Schatz, MD, FRCPC, and Terence J. Colgan, MD, FRCPC Department of Pathology, The Ibronto Hospital, 585 University Aenue, lbronto, Onta?-ioM5G 2C4, C m a d a
This case report details an osteogenic sarcoma arising in a vertebra in which cytokeratin intermediate filaments were detected immunohistochemically with three different antibodies. This feature was present not only in the primary neoplasm but also in t w o local recurrences and a metastasis t o the iliac bone. What is unique about this primary bone tumor, however, is the structural evidence for epithelial differentiation. Ultrastructurally, wellformed desmosomes and tonofilaments were present in all four surgically resected specimens. This tumor expands the list of soft tissue and bone tumors in which anomalous expression of intermediate filaments can occur but, more important, illustrates that changes in genetic expression of neoplasia of mesenchymal origin can result in paradoxic epithelial differentiation.
KEY WORDS. bone tumor, cytokeratin, immunocytochemtstry, sarcoma, ultrastructure. ~
INTRODUCTION When initially introduced t o the discipline of pathology, immunolabeling of the five intermediate filaments was heralded as the ideal method t o solve diagnostic problems. Each filament type seemed t o be restricted t o a specific tissue and/or cell type: cytokeratins t o the epithelia; vimentin t o mesenchymal tissues; desmin t o muscle; glial fibrillary acidic protein (GFAP) t o glia; and neurofilaments t o neurons. Tumors appeared t o emulate the expression of the particular intermediate filaments of their tissue of origin with a considerable degree of fidelity.’,’ The proliferation of polyclonal and monoclonal antibodies t o intermediate
The authors appreciate the assistance of Michael Stratis and the technologists at The Toronto Hospital Address correspondence to I Dardick
Copyright
~
~~
filaments that followed attested t o the widespread application o f these principles for diagnostic purposes. As immunohistochemical surveys for the complement of intermediate filaments in normal human tissues and their respective tumors accumulated, results began t o indicate that t w o or more of the classes of intermediate filaments could occur in a considerable variety of epithelial and mesenchymal cells, b o t h normal and neoplasCombinations of cytokeratins with either vimentin or GFAP for even both), desmin with cytokeratins, GFAP with vimentin, and cytokeratins with neurofilaments are not uncommon. A t times, dual expression of these polypeptides occurs even in individual tumor cell^.^,^ Biologic principles are frequently aberrant in neoplasia, so that it was not entirely unexpected that cytokeratins would be de-
Ultrastructural Pathology, 16:463-474, 1 9 9 2 1 9 9 2 by Hemisphere Publishing Corporation
cc>
~~-
463
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
464 tected in soft tissue tumors of different type^.^^'' What has been unusual is the seemingly purposeful search for soft tissue tumors with cytokeratin expression, a phenomenon recently addressed b y Swanson.” This has raised the issue of the reliability of the identification of intermediate filaments in these tumors. In the vast majority of such reports, there has been no structural evidence of epithelial differentiation. Only one malignant fibrous histiocytoma with cytokeratin detected in some tumor cells showed tonofilaments b y electron microscopy,12 and a chondrosarcoma has been reported with squamous differentiation.13 We report a case of osteogenic sarcoma of lumbar vertebral origin that also provides such evidence.
CLINICAL HISTORY In 1985, a 35-year-old man presented with recent onset of acute lower back pain accompanied by neurologic s ymptoms. He had a history of lower back pain, presumably secondary to a fall 2 years previously, controlled by nonsteroidal antiinflammatory drugs. Radiographs of the spine and subsequent computerized tomography (Fig. 1 ) revealed a destructive, calcifying lesion involving the first lumbar vertebra with extension of the tumor into the adjacent soft tissues. An en bloc resection was attempted with wound grafting with bone obtained from the left ilium. Histologically, the tumor was an osteogenic sarcoma. The margins of the resected specimen displayed malignant tumor, although follow-up X-ray studies did not disclose residual tumor. Investigation did not reveal metastatic tumor, and the patient received two courses of high-dose methotrexate and then adriam ycin, cisplatin, and bleom ycin. Although not detected on radiologic follow-up in 1988, recurrent tumor was discovered during surgical repair of a symptomatic vertebral pseudoarthrosis and was debulked. The patient received local radiation therapy and was well for 1 year ( 19891, when he again developed left-sided motor and sensory neurologic signs and symptoms. Lumbar radiographs
I. Dardick et a1
showed recurrent tumor extending from T- 1 1 to L -2, and tumor debulking lt ypical osteogenic sarcoma) was performed again. Two months later, follow-up disclosed metastatic osteogenic sarcoma in the left ilium which was treated by partial pelvectomy: In 199 1, clinical examination and radiography of the spine again revealed evidence of recurrent tumor at the L-5 level. Currently, the patient has minimal spinal cord function attributed to both recurrent tumor and radiation therapy to this region.
MATERIALS AND METHODS Representative tissue, which w a s both formalin fixed and snap frozen [in OCT medium (Miles Scientific, Naperville, IL) with liquid nitrogen], w a s available from the primary tumor ( 19 8 5 ) and the metastasis t o t h e pelvic bone ( 1 9 8 9 ) f o r routinely stained histologic sections and for immunocytochemistry. Only formalin-fixed, paraffin-embedded tumor tissue was available from the recurrences in 1 9 8 8 and 1 9 8 9 . All four specimens had tumor tissue fixed in glutaraldehyde and osmium tetroxide and embedded in Epon/Araldite for e I ec t r on mic r o s c o py. Immuno c y t o chemistry was performed on acetone-fixed frozen sections and sections c u t from the formalin-fixed tissues from the specimens w i t h both these materials available and only o n the formalin-fixed tissue from the other t w o specimens. The panel of antibodies used with their sources and dilutions is provided in Table 1. Frozen section immunocytochemistry was performed b y means of an indirect peroxidase t e ~ h n i q u e . ’ ~ Sections .’~ (6 pmi were air dried, fixed in acetone for 1 5 minutes, and then postfixed for 1 0 seconds in formol-calcium ( 100 m L 37% formaldehyde, 10 g calcium chloride, and 900 m L distilled w a t e r ) . l 5 For i m m u n o c y t o chemistry o n formalin-fixed and paraffinembedded tissues, a standard peroxidaseantiperoxidase technique w a s used. l 4 Tissue sections were exposed t o 2% fetal calf serum for 5 minutes t o inhibit nonspecific staining. Sections were treated with t w o changes o f 3% hydrogen peroxide in
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
Intriguing Case: Cytokeratins in Osteogenic Sarcoma
465
FIG. 1 Computed tomogram of the first lumbar vertebra partially destroyed by a calcifying tumor (open arrow) that extends into the adjacent soft tissues.
absolute methanol for 1 0 minutes each; this was followed by 3-minute washes in phosphate-buffered saline (PBS). Primary antibodies were applied for 1.5 hours at 37OC. Slides were then washed in PBS (three changes for 2 minutes each) before application of the secondary antibody [horseradish peroxidase-conjugated goat antibody t o mouse Fab', (Jackson Immunoresearch Laboratory, Inc., West Grove, PA; diluted 1 : 200)l for 1.5 hours
at room temperature. The colorimetric reaction was developed with 3,3' -diaminobenzidine (DAB) tetrahydrochloride/ hydrogen peroxide [ I 00 m g DAB t o 200 m L 0.05M tris (pH 7.6) plus 200 p L of 3 0 % hydrogen peroxide1 for 4 t o 5 minutes. Subsequently, the sections were counterstained with Myers hematoxylin. Typical examples of both epithelial and soft tissue tumors and of normal tissues served as positive controls for the immuno-
TABLE 1 Antibodies Used in the Study Antibodya Cytokeratins Epidermal (p, H M W ) AElIAE3 (ml C A M 5.2 ( m i Vimentin (m) GFAP Ip) Desmin (m) Carcinoembryonic antigen (p) Epithelial membrane antigen (m)
Source
Dilution
Dako, Santa Barbara, CA Hybritech, San Diego, CA Becton Dickinson, Mountain View, CA (further diluted) Biogenex, San Ramon, CA Dako Biogenex Dako Dako
1 : 200
1:50 1:4 1 :400 1 : 250 Prediluted 1 : 200 1 :40
Abbreviations: m, monoclonal antibody; p, polyclonal antibody; HMW, high molecular weight
466 staining. Irrelevant antibodies and substitution of buffer for primary antibodies served as negative controls. A tumor sample was only considered positively stained if individual cells were moderately t o strongly decorated ( 2 + t o 3 + / 3 + and if the staining reaction was confined t o the cytoplasm.
PATHOLOGY
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
Light Microscopy Histologically, each of the surgical specimens was a typical osteogenic sarcoma (Fig. 2). In addition t o osteoid (some of which was undergoing calcification), osteocytes, and many osteoblasts (Fig. 2a), there were extensive areas of osteoclasts admixed with mononuclear stromal cells (Fig. 2b). Other areas were formed almost exclusively of smaller, less differentiated tumor cells (Fig. 2b). Chondrocytic differentiation was not noted in either the primary neoplasm or any of the recurrences or the metastasis t o the ilium, and the histology did not differ in the specimens obtained after the patient underwent radiation therapy.
Electron Microscopy Electron microscopy was initially done per a soft tissue protocol in effect at Toronto General Hospital in 1985 and first detected the paradoxic findings described below. General features, the organization of the tumor cells, and noteworthy organelles were similar in the four surgical specimens. As illustrated from the recurrent tumor in 1989 (Fig. 3), the tumor cells were somewhat loosely organized and had irregularly contoured nuclei and a cytoplasm containing many profiles of rough endoplasmic reticulum. No basal lamina was developed, but collagen fibers and granulofibrillar deDosits were Dresent t o variable degrees between the tu.mor cells. In all the samples examined ultrastructurally, welldeveloped desmosomes complete w i t h projecting intermediate filaments (at times with a taillike shape) were readily identified (Fig. 4). Tonofilaments were not noted in
I. Dardick e t a1
the material available from the primary tumor, but these were seen in the other three specimens (Figs. 3 and 4b), often in a perinuclear pattern (Fig. 3 ) .
Immunocytochemistry Results of immunostaining with the panel of selected antibodies on both formalinfixed tissues and frozen sections are provided in Table 2. There were differences in the proportion of tumor cells staining for cytokeratin polypeptides with frozen and formalin-fixed sections, which may reflect both the selection of different regions of the tumor and the effects of the fixative. Cytokeratin-positive tumor cells tended t o be focally grouped (Fig. 5), and few t o many were intermingled among negative cells (Fig. 5, a t o d); osteoclastlike giant cells were never positive with any of the three antibodies t o cytokeratin (Fig. 5, b and d). Some cytokeratin-positive osteocytes were present within osteoid (Fig. 5, a and d). In all specimens, variable numbers of tumor cells were stained with a polyclonal antibody t o high-molecular weight cytokeratins (Fig. 5, a and b; Table 2). In fact, the highest proportion of tumor cells positive for cytokeratins ( 5 0 % t o 6 0 % ) occurred on staining of frozen sections from the 1989 metastasis t o the pelvic bone with this antibody (Table 2). The degree of detection of vimentin was fixation dependent, but staining occurred more or less in all samples (Fig. 5e). lmmunostaining for desmin was negative. GFAP was detected in a small number of tumor cells only in the primary tumor. Carcinoembryonic antigen also was noted only in the primary tumor ( 5 % t o 1 0 % of tumor cells), but a small proportion of tumor cells expressed epithelial membrane antigen in all four surgical specimens, although this was most evident in the frozen sections from the 1989 specimen.
DISCUSSION Detection of cytokeratin in nonepithelial tissue and tumors, as for its counterpart vimentin, is an increasingly observed phe-
467
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
Intriguing Case: Cytokeratins in Osteogenic Sarcoma
FIG. 2
Osteogenic sarcoma. (a) Representative histology from the original vertebral tumor (1985) with extensive osteoid, some of which is undergoing calcification. Hematoxylin and eosin IH&E), x 160. (b) Histology of the metastatic tumor (pelvic bone) in 1989 with osteoid and associated osteoblasts, many osteoclasts, and zones of undifferentiated tumor cells (curved arrow). H&E, x 160.
I. Dardick et a1
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
468
FIG. 3 Surve y-type electron micrograph showing roundish, slightly separated tumor cells with rough endoplasmic reticulum, perinuclear tono filaments (arrows), and occasional intercellular junctions (arrowheads). Urany l acetate and lead citrate, x 5,400.
469
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
Intriguing Case: Cytokeratins in Osteogenic Sarcoma
FIG. 4 Intermediate filament-associated intercellular junctions (arrows) with dense plaques aQacent to the plasmalemma but no intermediate line. (a) The original resected tumor 11985). Uranyl acetate and lead citrate, x 40,000. fb) The 1989 metastasis to the pelvic bone (ilium) with tonofilaments (TI. Uranyl acetate and lead citrate, x 30,000.
TABLE 2 Percentage of Tumor Cells lmmunostained in the Osteogenic Sarcoma Primary 1985 Antibody Cytokeratins HMWa AEl/AE3 CAM 5.2 Vimentin GFAP Desmin Carcinoembryonic antigen Epithelial membrane antigen
Frozen
Formalin
Recurrence 1988 (formalin)
10-15 10-15 20-25 100 5-10 Negative
10-15 30-40 Negative 50-60 Negative Negative
40-50 30-40 Negative 50 Negative Negative
10-15 30-40 Negative 30-35 Negative Negative
50-60 -5
ISSN: 0191-3123 (Print) 1521-0758 (Online) Journal homepage: http://www.tandfonline.com/loi/iusp20
Osteogenic Sarcoma with Epithelial Differentiation Irving Dardick, Julie E. Schatz & Terence J. Colgan To cite this article: Irving Dardick, Julie E. Schatz & Terence J. Colgan (1992) Osteogenic Sarcoma with Epithelial Differentiation, Ultrastructural Pathology, 16:4, 463-474, DOI: 10.3109/01913129209057831 To link to this article: http://dx.doi.org/10.3109/01913129209057831
Published online: 10 Jul 2009.
Submit your article to this journal
Article views: 2
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iusp20 Download by: [Universität Osnabrueck]
Date: 15 March 2016, At: 12:52
Intriguing Case Osteogenic Sarcoma with Epithelial Differentiation _______
~
_
_
.
_
Irving Dardick, MD, FRCPC Department of Pathology, University of lbronto, Banting Institute, 100 College Street, Ibronto, Ontario M5G lL5, Canada
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
Julie E. Schatz, MD, FRCPC, and Terence J. Colgan, MD, FRCPC Department of Pathology, The Ibronto Hospital, 585 University Aenue, lbronto, Onta?-ioM5G 2C4, C m a d a
This case report details an osteogenic sarcoma arising in a vertebra in which cytokeratin intermediate filaments were detected immunohistochemically with three different antibodies. This feature was present not only in the primary neoplasm but also in t w o local recurrences and a metastasis t o the iliac bone. What is unique about this primary bone tumor, however, is the structural evidence for epithelial differentiation. Ultrastructurally, wellformed desmosomes and tonofilaments were present in all four surgically resected specimens. This tumor expands the list of soft tissue and bone tumors in which anomalous expression of intermediate filaments can occur but, more important, illustrates that changes in genetic expression of neoplasia of mesenchymal origin can result in paradoxic epithelial differentiation.
KEY WORDS. bone tumor, cytokeratin, immunocytochemtstry, sarcoma, ultrastructure. ~
INTRODUCTION When initially introduced t o the discipline of pathology, immunolabeling of the five intermediate filaments was heralded as the ideal method t o solve diagnostic problems. Each filament type seemed t o be restricted t o a specific tissue and/or cell type: cytokeratins t o the epithelia; vimentin t o mesenchymal tissues; desmin t o muscle; glial fibrillary acidic protein (GFAP) t o glia; and neurofilaments t o neurons. Tumors appeared t o emulate the expression of the particular intermediate filaments of their tissue of origin with a considerable degree of fidelity.’,’ The proliferation of polyclonal and monoclonal antibodies t o intermediate
The authors appreciate the assistance of Michael Stratis and the technologists at The Toronto Hospital Address correspondence to I Dardick
Copyright
~
~~
filaments that followed attested t o the widespread application o f these principles for diagnostic purposes. As immunohistochemical surveys for the complement of intermediate filaments in normal human tissues and their respective tumors accumulated, results began t o indicate that t w o or more of the classes of intermediate filaments could occur in a considerable variety of epithelial and mesenchymal cells, b o t h normal and neoplasCombinations of cytokeratins with either vimentin or GFAP for even both), desmin with cytokeratins, GFAP with vimentin, and cytokeratins with neurofilaments are not uncommon. A t times, dual expression of these polypeptides occurs even in individual tumor cell^.^,^ Biologic principles are frequently aberrant in neoplasia, so that it was not entirely unexpected that cytokeratins would be de-
Ultrastructural Pathology, 16:463-474, 1 9 9 2 1 9 9 2 by Hemisphere Publishing Corporation
cc>
~~-
463
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
464 tected in soft tissue tumors of different type^.^^'' What has been unusual is the seemingly purposeful search for soft tissue tumors with cytokeratin expression, a phenomenon recently addressed b y Swanson.” This has raised the issue of the reliability of the identification of intermediate filaments in these tumors. In the vast majority of such reports, there has been no structural evidence of epithelial differentiation. Only one malignant fibrous histiocytoma with cytokeratin detected in some tumor cells showed tonofilaments b y electron microscopy,12 and a chondrosarcoma has been reported with squamous differentiation.13 We report a case of osteogenic sarcoma of lumbar vertebral origin that also provides such evidence.
CLINICAL HISTORY In 1985, a 35-year-old man presented with recent onset of acute lower back pain accompanied by neurologic s ymptoms. He had a history of lower back pain, presumably secondary to a fall 2 years previously, controlled by nonsteroidal antiinflammatory drugs. Radiographs of the spine and subsequent computerized tomography (Fig. 1 ) revealed a destructive, calcifying lesion involving the first lumbar vertebra with extension of the tumor into the adjacent soft tissues. An en bloc resection was attempted with wound grafting with bone obtained from the left ilium. Histologically, the tumor was an osteogenic sarcoma. The margins of the resected specimen displayed malignant tumor, although follow-up X-ray studies did not disclose residual tumor. Investigation did not reveal metastatic tumor, and the patient received two courses of high-dose methotrexate and then adriam ycin, cisplatin, and bleom ycin. Although not detected on radiologic follow-up in 1988, recurrent tumor was discovered during surgical repair of a symptomatic vertebral pseudoarthrosis and was debulked. The patient received local radiation therapy and was well for 1 year ( 19891, when he again developed left-sided motor and sensory neurologic signs and symptoms. Lumbar radiographs
I. Dardick et a1
showed recurrent tumor extending from T- 1 1 to L -2, and tumor debulking lt ypical osteogenic sarcoma) was performed again. Two months later, follow-up disclosed metastatic osteogenic sarcoma in the left ilium which was treated by partial pelvectomy: In 199 1, clinical examination and radiography of the spine again revealed evidence of recurrent tumor at the L-5 level. Currently, the patient has minimal spinal cord function attributed to both recurrent tumor and radiation therapy to this region.
MATERIALS AND METHODS Representative tissue, which w a s both formalin fixed and snap frozen [in OCT medium (Miles Scientific, Naperville, IL) with liquid nitrogen], w a s available from the primary tumor ( 19 8 5 ) and the metastasis t o t h e pelvic bone ( 1 9 8 9 ) f o r routinely stained histologic sections and for immunocytochemistry. Only formalin-fixed, paraffin-embedded tumor tissue was available from the recurrences in 1 9 8 8 and 1 9 8 9 . All four specimens had tumor tissue fixed in glutaraldehyde and osmium tetroxide and embedded in Epon/Araldite for e I ec t r on mic r o s c o py. Immuno c y t o chemistry was performed on acetone-fixed frozen sections and sections c u t from the formalin-fixed tissues from the specimens w i t h both these materials available and only o n the formalin-fixed tissue from the other t w o specimens. The panel of antibodies used with their sources and dilutions is provided in Table 1. Frozen section immunocytochemistry was performed b y means of an indirect peroxidase t e ~ h n i q u e . ’ ~ Sections .’~ (6 pmi were air dried, fixed in acetone for 1 5 minutes, and then postfixed for 1 0 seconds in formol-calcium ( 100 m L 37% formaldehyde, 10 g calcium chloride, and 900 m L distilled w a t e r ) . l 5 For i m m u n o c y t o chemistry o n formalin-fixed and paraffinembedded tissues, a standard peroxidaseantiperoxidase technique w a s used. l 4 Tissue sections were exposed t o 2% fetal calf serum for 5 minutes t o inhibit nonspecific staining. Sections were treated with t w o changes o f 3% hydrogen peroxide in
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
Intriguing Case: Cytokeratins in Osteogenic Sarcoma
465
FIG. 1 Computed tomogram of the first lumbar vertebra partially destroyed by a calcifying tumor (open arrow) that extends into the adjacent soft tissues.
absolute methanol for 1 0 minutes each; this was followed by 3-minute washes in phosphate-buffered saline (PBS). Primary antibodies were applied for 1.5 hours at 37OC. Slides were then washed in PBS (three changes for 2 minutes each) before application of the secondary antibody [horseradish peroxidase-conjugated goat antibody t o mouse Fab', (Jackson Immunoresearch Laboratory, Inc., West Grove, PA; diluted 1 : 200)l for 1.5 hours
at room temperature. The colorimetric reaction was developed with 3,3' -diaminobenzidine (DAB) tetrahydrochloride/ hydrogen peroxide [ I 00 m g DAB t o 200 m L 0.05M tris (pH 7.6) plus 200 p L of 3 0 % hydrogen peroxide1 for 4 t o 5 minutes. Subsequently, the sections were counterstained with Myers hematoxylin. Typical examples of both epithelial and soft tissue tumors and of normal tissues served as positive controls for the immuno-
TABLE 1 Antibodies Used in the Study Antibodya Cytokeratins Epidermal (p, H M W ) AElIAE3 (ml C A M 5.2 ( m i Vimentin (m) GFAP Ip) Desmin (m) Carcinoembryonic antigen (p) Epithelial membrane antigen (m)
Source
Dilution
Dako, Santa Barbara, CA Hybritech, San Diego, CA Becton Dickinson, Mountain View, CA (further diluted) Biogenex, San Ramon, CA Dako Biogenex Dako Dako
1 : 200
1:50 1:4 1 :400 1 : 250 Prediluted 1 : 200 1 :40
Abbreviations: m, monoclonal antibody; p, polyclonal antibody; HMW, high molecular weight
466 staining. Irrelevant antibodies and substitution of buffer for primary antibodies served as negative controls. A tumor sample was only considered positively stained if individual cells were moderately t o strongly decorated ( 2 + t o 3 + / 3 + and if the staining reaction was confined t o the cytoplasm.
PATHOLOGY
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
Light Microscopy Histologically, each of the surgical specimens was a typical osteogenic sarcoma (Fig. 2). In addition t o osteoid (some of which was undergoing calcification), osteocytes, and many osteoblasts (Fig. 2a), there were extensive areas of osteoclasts admixed with mononuclear stromal cells (Fig. 2b). Other areas were formed almost exclusively of smaller, less differentiated tumor cells (Fig. 2b). Chondrocytic differentiation was not noted in either the primary neoplasm or any of the recurrences or the metastasis t o the ilium, and the histology did not differ in the specimens obtained after the patient underwent radiation therapy.
Electron Microscopy Electron microscopy was initially done per a soft tissue protocol in effect at Toronto General Hospital in 1985 and first detected the paradoxic findings described below. General features, the organization of the tumor cells, and noteworthy organelles were similar in the four surgical specimens. As illustrated from the recurrent tumor in 1989 (Fig. 3), the tumor cells were somewhat loosely organized and had irregularly contoured nuclei and a cytoplasm containing many profiles of rough endoplasmic reticulum. No basal lamina was developed, but collagen fibers and granulofibrillar deDosits were Dresent t o variable degrees between the tu.mor cells. In all the samples examined ultrastructurally, welldeveloped desmosomes complete w i t h projecting intermediate filaments (at times with a taillike shape) were readily identified (Fig. 4). Tonofilaments were not noted in
I. Dardick e t a1
the material available from the primary tumor, but these were seen in the other three specimens (Figs. 3 and 4b), often in a perinuclear pattern (Fig. 3 ) .
Immunocytochemistry Results of immunostaining with the panel of selected antibodies on both formalinfixed tissues and frozen sections are provided in Table 2. There were differences in the proportion of tumor cells staining for cytokeratin polypeptides with frozen and formalin-fixed sections, which may reflect both the selection of different regions of the tumor and the effects of the fixative. Cytokeratin-positive tumor cells tended t o be focally grouped (Fig. 5), and few t o many were intermingled among negative cells (Fig. 5, a t o d); osteoclastlike giant cells were never positive with any of the three antibodies t o cytokeratin (Fig. 5, b and d). Some cytokeratin-positive osteocytes were present within osteoid (Fig. 5, a and d). In all specimens, variable numbers of tumor cells were stained with a polyclonal antibody t o high-molecular weight cytokeratins (Fig. 5, a and b; Table 2). In fact, the highest proportion of tumor cells positive for cytokeratins ( 5 0 % t o 6 0 % ) occurred on staining of frozen sections from the 1989 metastasis t o the pelvic bone with this antibody (Table 2). The degree of detection of vimentin was fixation dependent, but staining occurred more or less in all samples (Fig. 5e). lmmunostaining for desmin was negative. GFAP was detected in a small number of tumor cells only in the primary tumor. Carcinoembryonic antigen also was noted only in the primary tumor ( 5 % t o 1 0 % of tumor cells), but a small proportion of tumor cells expressed epithelial membrane antigen in all four surgical specimens, although this was most evident in the frozen sections from the 1989 specimen.
DISCUSSION Detection of cytokeratin in nonepithelial tissue and tumors, as for its counterpart vimentin, is an increasingly observed phe-
467
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
Intriguing Case: Cytokeratins in Osteogenic Sarcoma
FIG. 2
Osteogenic sarcoma. (a) Representative histology from the original vertebral tumor (1985) with extensive osteoid, some of which is undergoing calcification. Hematoxylin and eosin IH&E), x 160. (b) Histology of the metastatic tumor (pelvic bone) in 1989 with osteoid and associated osteoblasts, many osteoclasts, and zones of undifferentiated tumor cells (curved arrow). H&E, x 160.
I. Dardick et a1
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
468
FIG. 3 Surve y-type electron micrograph showing roundish, slightly separated tumor cells with rough endoplasmic reticulum, perinuclear tono filaments (arrows), and occasional intercellular junctions (arrowheads). Urany l acetate and lead citrate, x 5,400.
469
Downloaded by [Universität Osnabrueck] at 12:52 15 March 2016
Intriguing Case: Cytokeratins in Osteogenic Sarcoma
FIG. 4 Intermediate filament-associated intercellular junctions (arrows) with dense plaques aQacent to the plasmalemma but no intermediate line. (a) The original resected tumor 11985). Uranyl acetate and lead citrate, x 40,000. fb) The 1989 metastasis to the pelvic bone (ilium) with tonofilaments (TI. Uranyl acetate and lead citrate, x 30,000.
TABLE 2 Percentage of Tumor Cells lmmunostained in the Osteogenic Sarcoma Primary 1985 Antibody Cytokeratins HMWa AEl/AE3 CAM 5.2 Vimentin GFAP Desmin Carcinoembryonic antigen Epithelial membrane antigen
Frozen
Formalin
Recurrence 1988 (formalin)
10-15 10-15 20-25 100 5-10 Negative
10-15 30-40 Negative 50-60 Negative Negative
40-50 30-40 Negative 50 Negative Negative
10-15 30-40 Negative 30-35 Negative Negative
50-60 -5
