Spindle Cell Carcinoma of the Conjunctiva An Immunohistochemical and Ultrastructural Study of Six Cases ANNE C. HUNTINGTON, MD,t JOHN M. LANGLOSS, DVM, PhD/ AHMED A. HIDAYAT, MD3
Abstract: Six cases of conjunctival spindle cell carcinoma, a rare variant of squamous cell carcinoma, were studied. The median age of the three men and three women was 63.5 years. The tumors appeared as a single nodule in some patients or diffusely involved the conjunctiva in others. Two of the four individuals with intraocular extension presented with phthisis bulbi. Polyclonal anti keratin antibody was helpful and gave the most consistent results when compared with monoclonal anti keratin antibodies, AE1/3 and PKK1. The electron microscopic study of four lesions also established the epithelial nature of the tumor cells. Intracytoplasmic tonofilaments and a few desmosomes were present. Histopathologically, this variant of squamous cell carcinoma is difficult to distinguish from other spindle cell tumors, and this study demonstrates the value of immunohistochemistry and electron microscopy in supporting the correct diagnosis. Ophthalmology 1990; 97:711-717
Spindle cell carcinoma, a rare variant of squamous cell carcinoma, occurs in skin l - 3 and mucous membranes. 2,3 A review ofthe literature showed only three reported cases of spindle cell carcinoma involving the conjunctiva,4,5 and in one of these cases electron microscopy confirmed the diagnosis. Histopathologically, spindle cell carcinomas are difficult to distinguish from other spindle cell tumors, inOriginally received: October 2, 1989. Revision accepted: January 15, 1990. Department of Ophthalmology and Visual Sciences, Kentucky Lions Research Institute, University of Louisville, Louisville. 2 Drug Safety Division, Porer Central Research, Fort Washington. 3 Department of Ophthalmic Pathology, Armed Forces Institute of Pathol· ogy, Washington, DC. 1
Presented at the Association for Research in Vision and Ophthalmology Annual Meeting, Sarasota, May 1, 1985. The opinions and assertions contained herein are the private views of the authors and should not be construed as being official or representing the views of the Department of the Army or the Department of Defense. Reprint requests to Ahmed A. Hidayat, MD, Department of Ophthalmic Pathology, Armed Forces Institute of Pathology, Washington, DC 20306.
cluding amelanotic melanoma, malignant schwannoma, fibrosarcoma, leiomyosarcoma, and malignant fibrous histiocytoma. In the past, considerable reliance has been placed on the ultrastructural demonstration of epithelial differentiation to identify spindle cell carcinoma from other spindle cell tumors. 2- 6 Interpretation may be difficult,7 however, where the characteristic features are not always demonstrable ultrastructurally because of small samplings/,8 and the procedure is costly and time-consuming. More recently, immunohistochemistry has been used to aid in the classification of tumors in those cases in which morphologic features alone are insufficient for diagnosis. 9 ,lo We undertook this immunohistochemical study to determine the value of keratin and epithelial membrane antigen (EMA) as markers in identifying the spindle cell variant of squamous cell carcinoma of the conjunctiva. Recent studies have shown that the immunohistochemical detection of keratin 11-17 and EMA I8 - 20 offers a powerful tool for identifying tumors of epithelial origin. These markers are particularly valuable in recognizing spindle cell carcinoma and distinguishing it from other spindle cell tumors. Electron microscopy also is useful in establishing epithelial differentiation, and 711
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Table 1. Clinical Data for Six Patients with Spindle Cell Carcinoma of Conjunctiva Patient No. Sex/Age (yrs)
Clinical Appearance
Intraocular Extension
Treatment
1 F/30 2 Mj72 3 M/55 4 M/85 5 F/80 6 F/50
Unknown Limbal papillomas, phthisis bulbi Multiple conjunctival nodules, phthiSis bulbi Single limbal mass overlapping cornea Single nodule of bulbar conjunctiva Single nodule of bulbar conjunctiva
Yes Yes Yes No Yes No
Exenteration Enucleation Enucleation Enucleation Exenteration Excision
four of the six neoplasms in this study were studied ultrastructurally.
MATERIALS AND METHODS Twenty-four cases diagnosed as spindle cell tumor of the conjunctiva on file in the Registry of Ophthalmic Pathology at the Armed Forces Institute of Pathology were reviewed. Of these, only six had sufficient material (wet tissue, blocks, and/or unstained sections) for inclusion in this study. Hematoxylin-eosin-stained sections and special stains for melanin (either Fontana and/or Warthin-Starry at pH 3.3) were available for all cases. Five-micron glued sections offormalin-fixed, paraffin-embedded tissue were prepared for immunohistochemical studies. In four cases, 4% formaldehyde-fixed tissue was processed for electron microscopic studies. 21 Antibodies used for immunohistochemical studies were obtained in the following methods. Polyclonal antikeratin antiserum was derived in rabbits using keratin proteins isolated from human plantar calluses according to the method of Sun and Green. 22 The antibodies were affinity isolated using purified keratin proteins linked to cyanogen bromide-activated sepharose 4B (Pharmacia Chemical Co, Piscataway, NJ). The monoclonal keratin antibodies, PKKI (Labsystems-USA, Chicago, IL) and AEl/3 (Hybritech, San Diego, CA) were obtained from commercial sources. The monoclonal anti-EMA antibody also was obtained from a commercial source (Dako Laboratories, Copenhagen, Denmark; US Distributor, Accurate Chemical and Scientific Co, Westbury, NY). In an attempt to exclude other spindle cell tumors, two additional antibodies were used in this study: anti-S-lOO protein and antidesmin. Immunostaining for immunoreactive keratin, S-lOO protein, and desmin using polyclonal antibodies was performed as in previously described procedures. 23 The peroxidase antiperoxidase (PAP) method introduced by Sternberger4 was used. For the demonstration of keratin and desmin, sections were predigested with 0.1 % bovine trypsin (Sigma Chemical Co, St. Louis, MO) in 0.1M phosphate buffer, pH 7.8, at 37°C for 15 or 60 minutes. To reduce background staining, sections were incubated overnight in 5% normal swine serum. Sections were then incubated with appropriate primary antibodies at room temperature for 30 minutes, followed by 30-minute incubations with excess bridge antibody (swine anti-rabbit 712
Igs) and rabbit PAP complexes (Dako Laboratories, Copenhagen, Denmark) with intervening washes in O.OI-M phosphate-buffered saline, pH 7.4. Sections were developed in a solution containing 0.05% H 20 2 and 0.016% diaminobenzadine tetrahydrochloride (Sigma Chemical Co, St. Louis, MO) and counterstained with hematoxylin. Duplicate sections immunostained as above, except for the substitution of normal rabbit serum for the primary antibody, served as negative controls. For the monoclonal antibodies the avidin-biotin complex method (Vector Laboratories, Burlingame, CA) introduced by Hsu 25 was used. Predigested sections were incubated overnight in 5% horse serum and then incubated successively for 30 minutes at room temperature in monoclonal antibodies, biotinylated horse antimouse immunoglobulin (Vector Laboratories, Burlingame, CA), and avidin-biotin complex, with intervening washes in phosphate-buffered saline. Development of the chromogen and counterstaining was completed as described for the PAP method. Normal mouse serum was used as a negative control. In both the PAP and avidin-biotin complex techniques, uninvolved squamous epithelium served as an intrinsic positive control for the keratins and EMA.
RESULTS CLINICAL FINDINGS
The clinical data are summarized in Table 1. The median age ofthe six patients at the time of surgery was 63.5 years (range, 30-85 years). Three patients were male and three were female. The tumors originated in the conjunctiva as single or multiple nodules that were either located at the limbus or diffusely involved the conjunctiva more extensively. One tumor in our series (case 4) was a large sessile limbal mass that overlapped the cornea and obscured vision (Fig 1), and two others (cases 5 and 6) had single nodules of the bulbar conjunctiva. In the other two patients, the conjunctiva was more diffusely involved, and in case 3 the entire conjunctival sac was studded with small tumor nodules. The corneoscleral area was invaded to a varying extent in all cases. In four patients, there was intraocular extension, and in two of these (cases 2 and 3) the tumors were predominantly intraocular and the clinical diagnosis was phthisis bulbi. Both of the patients with phthisis bulbi had protracted and complicated ocular histories, which diverted attention from the conjunctival
Fig 1. Top left, a large and fleshy conjunctival mass in the right eye overlaps a scarred and vascularized cornea, Courtesy of Professor J. S. Jain (AFIP Neg. 86-5207). Fig 2. Second row left, diffuse conjunctival tumor that has invaded the limbus. Perforation of a corneal ulcer is present (hematoxylin-eosin; original magnification, X3.5, AFIP Neg. 86-5231). Fig 3. Top right, large intraocular mass involves the vitreous and detached retina (hematoxylineosin; original magnification, X3.5, AFIP Neg. 86-5221). Fig 4. Third row left, limbal epithelium shows acanthosis and loss of polarity of the abnormal cells that extend to the surface. These pathologic changes are consistent with carcinoma in situ (hematoxylin-eosin; original magnification, X300, AFIP Neg. 865219). Fig 5. Third row right, hypercellular tumor is composed of spindle-shaped cells, arranged in interlacing fascicles. The tumor resembles fibrosarcoma or fibrous histiocytoma (hematoxylineosin; original magnification, X75, AFIP Neg. 86-5229). Fig 6. Fourth row left, atypical spindle-shaped cells have elongated and vesicular nuclei with irregular nuclear membrane and prominent nucleoli. Abnormal mitotic figures are present (hematoxylin-eosin; original magnification, X630, AFIP Neg. 865233). Fig 7. Bottom right, some of the pleomorphic and bizarre tumor cells have hyperchromatic nuclei and dense eosinophilic cytoplasm. The stroma is infiltrated by inflammatory cells (hematoxylin-eosin; original magnification, X630, AFIP Neg. 86-5220). Fig 8. Bottom left, spindle-shaped tumor cells show positive staining for polyclonal antikeratin. The loose and edematous stroma is infiltrated by inflammatory cells (peroxidase-antiperoxidase technique; original magnification, X450, AFIP Neg. 86-5223).
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Table 2. Immunohistochemical Results of Anti-epithelial Antibodies Cases
Polyclonal anti keratin AE1/3 PKK1 Anti-EMA
+ +
2
3
4
+ + +
+
+
5
6
+ +
pathology. In case 2, the patient had a 9-year history of recurrent herpetic keratitis for which multiple intraocular surgical procedures had been undertaken. Conjunctival papillomas had been noted 3 years before enucleation but a biopsy had been done. It is likely that the surgical procedures, which included three penetrating keratoplasties, cataract extraction, and vitrectomy, contributed to the intraocular spread of the tumor. The other patient with a phthisical eye (case 3) had a history of unspecified ocular inflammation 40 years before enucleation. The entire conjunctival sac in this case was extensively thickened and distorted by many rounded nodules. All patients had been treated surgically either by local excision ofthe tumor, enucleation, or exenteration. Follow-up data were incomplete because some patients were new and others were lost to follow-up.
Fig 9. A desmosome (arrow) between tumor cells is present. The nuclear membrane is irregular (original magnification, X35,000, AFIP Neg. 869160).
LIGHT MICROSCOPIC FINDINGS
The tumors appeared as a single polypoid limbal mass or with diffuse involvement of the entire bulbar conjunctiva (Fig 2). In one lesion, both the bulbar and tarsal conjunctivae were invaded by the tumor in the form of multiple small nodules. In one phthisical eye that had undergone multiple surgical procedures, including keratoplasty, before enucleation the large intraocular tumor involved the vitreous and detached gliotic retina (Fig 3). In that eye, the limbal epithelium showed abnormal changes consistent with carcinoma in situ (Fig 4), and serial sections showed invasion of the transplanted corneal stroma by atypical spindle-shaped cells. The tumors were composed of spindle-shaped cells arranged haphazardly in interlacing fascicles (Fig 5) or were widely separated by edematous-appearing stroma containing many chronic inflammatory cells. The nuclei of the tumor cells were mostly elongated and vesicular with an irregular nuclear membrane and prominent nucleoli (Figs 6, 7). Few pleomorphic and bizarre cells with hyperchromatic nuclei and dense eosinophilic cytoplasm also were present (Fig 7). Mitotic figures were frequent (Figs 6, 7). Continuity between the tumor and the overlying epithelium was noted in four lesions. In the remaining two lesions, there was surface ulceration. Special stains for melanin (Warthin-Starry and Fontana) were negative in all tumors. IMMUNOHISTOCHEMICAL STUDIES
The immunohistochemical results are summarized in Table 2. Immunohistochemical staining for epithelial an714
tigens was successful in four of the six tumors (cases 14). There was some variation in the staining intensity, but positive cells were readily recognized by the characteristic gold-brown to red-brown staining product. The polyclonal antikeratin antibody decorated cells in four lesions (Fig 8). Identifiable keratin was focal in three of these tumors and diffuse in one. Of the monoclonal antibodies, PKK 1decorated cells in two tumors, anti-EMA in two, and AE1/ 3 in one. Immunostaining with the monoclonal antibodies resulted in focal, less-intense staining than with the polyclonal antikeratin antibody. Uninvolved conjunctival epithelium served as an intrinsic control for epithelial antigens and was positive in all six cases. The immunologic markers S-1 00 protein and desmin were not demonstrated in any of the lesions studied. ELECTRON MICROSCOPIC STUDIES
Tumor cells were predominantly spindle shaped with elongated nuclei and irregular nuclear membranes. A few desmosomes were present (Fig 9) between adjacent cells, but in many areas where the cells were separated from each other by edematous stroma the desmosomes were absent. The cytoplasm contained rough-surfaced endoplasmic reticulum, few mitochondria, and variable amounts of to no filaments (Fig 10). In addition to the tumor cells, there was evidence of reactive stromal proliferation where fibroblasts were seen in the conjunctival lesions and glial cells were present adjacent to the tumor cells in the involved retina.
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Fig 10. The cytoplasm contains many bundles of tonofilaments (T) and rough-surfaced endoplasmic reticulum (original magnification. X21,OOO, AFIP Neg. 9162).
DISCUSSION In this report, the median age of the patients at the time of surgery was 63.5 years and the sex incidence was equal. The tumor usually appears clinically as single or multiple nodules located at the limbus, as in previous reports,4,5 but in some patients it may involve the conjunctiva more diffusely. It is interesting that two of the patients in our study (cases 2 and 3) had conjunctival papillomas and later developed phthisis bulbi. A primary conjunctival tumor with intraocular extension was an unsuspected finding. Histopathologically, the interpretation of the intraocular findings in these two cases was extremely difficult. In one lesion (case 2), the main part of a cellular mass was intimately related to a detached and gliotic retina, and the differential diagnosis was between massive retinal gliosis and adenocarcinoma of the retinal pigment epithelium. In the other case of phthisis bulbi (case 3), adenocarcinoma of the nonpigmented ciliary epithelium was the initial diagnosis. The differential diagnoses in the other four patients included amelanotic melanoma, Kaposi's sarcoma, leiomyosarcoma, and malignant schwan noma, in addition to spindle cell carcinoma. Although conclusions on tumor behavior cannot be drawn from this study because of the lack of follow-up data, the fact that three patients required enucleation and two required exenteration is indicative of local aggressiveness. This is in agreement with other studies,4,5 and a
wide excision of the initial lesion is recommended to avoid recurrences. Before the application of immunohistochemical techniques to tumor diagnosis, the diagnosis of spindle cell carcinoma rested on characteristic histologic and ultrastructural features. The most consistent criterion for histologic diagnosis is continuity of the spindle cell tumor with the surface epithelium, although a careful search through numerous sections is often required to demonstrate this feature. Cellular atypia and even frank malignant change within the overlying epithelium also may be present. The observation of both these changes is dependent on an intact surface epithelium. Unfortunately, surface ulceration with loss of these valuable clues is a common feature of this neoplasm. In this study, the conjunctiva in case 2 showed changes consistent with carcinoma in situ. Transition from spindle to squamous cells may be seen, but this is an inconsistent feature and was not observed in any of our cases. The diagnosis of spindle cell carcinoma may be reliably made by the ultrastructural presence of to no filaments and desmosomes, although extensive sampling is often needed to demonstrate these structures. 6 ,26 The recent literature contains several reports that indicate that the cytoplasmic keratins ll - 17 and epithelial membrane antigen l8 - 2o are reliable diagnostic markers for tumors of epithelial origin. The keratins, 16,27.28 which are present in almost all epithelial cells, are a group of at least 19 closely related polypeptides. They form one of the five 715
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classes of cytoskeletal intermediate filament proteins that are immunologically and biochemically distinct. 27,29 Many investigators have reported that the filaments of neoplastic cells mirror those of the cell of tumor origin,13,14,30 and the development of specific antisera to intermediate filament proteins has become a useful adjunct to distinguish morphologically similar tumors, Keratins have been demonstrated in a number of tumors that affect the ocular adnexa, including basal cell,1l,13,15,17,31 squamous cell 11,13,15,17,31 sebaceous cell 17 and mucoepidermoid carcin~mas, 17 Epithelial membrane antigen, or milk fat globule membrane antigen, is a large molecular weight glycoconjugate that also is widely distributed in normal epithelia and tumors derived from them. 12,18,20,32 Although EMA is not always demonstrable in normal squamous epithelium, alteration by either nonneoplastic or neoplastic disease processes may result in its expression. 19 There is recent evidence that expression of EMA is not restricted to epithelial cells but also may be demonstrated on reactive and neoplastic plasma cells and some lymphomas. 33 In this study, the polyclonal antikeratin antibody was helpful and gave the most consistent results with positive staining of cells in four tumors, whereas the monoclonal antikeratin antibodies, AEl/3 and PKKl, were positive in only one and two tumors respectively. The polyclonal antiepidermal keratin antibody is the antikeratin that has been used most frequently in similar studies. The monoclonal antikeratin antibodies are a more recent introduction. The antikeratin AEl/3 is composed of monoclonal antibodies AEI and AE3, which recognize most acidic and basic keratin proteins. 16,34 The other monoclonal antikeratin we used was PKK1, a monoclonal antibody raised against a pig kidney epithelial cell line, which is directed against low molecular weight keratin proteins found principally in simple epithelia. 35 ,36 In a study on renal carcinoma, PKK 1, used in conjunction with a polyclonal antikeratin antibody, provided valuable information on the cellular origin and differentiation of these tumors.35 In another study, PKKI was found to be less helpful than the polyclonal antikeratin antibody and the monoclonal mixture, AEI/3. 37 We found only two of the six tumors stained with the monoclonal anti-EMA antibody. Although it has been reported that squamous epithelium does not express EMA,19 we did not find this to be true because EMA was demonstrated in the overlying squamous epithelium in all six tumors included in this study. Immunohistochemistry has a number of advantages over electron microscopy. A larger area of tumor can be examined, thus avoiding the introduction of error because of inadequate sampling in ultrastructural studies. In electron microscopy, several blocks of tumor tissue often have to be examined to find tonofilaments and desmosomes, the characteristic ultrastructural features of epithelial cells, because stromal cells may be indistinguishable from tumor cells in all other respects. Tonofilaments are the ultrastructural equivalent of keratin, and the focal expression of keratin, demonstrated immunohistochemically in the keratin-positive tumors, correlates with the sparsity oftu716
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mor cells with ultrastructural evidence of squamous differentiation reported by various authors. 7,26 Immunohistochemistry also is less time-consuming than electron microscopy. One disadvantage of immunohistochemistry is that the fixation method influences the sensitivity of the technique. Optimal results with immunohistochemistry are achieved by using either unfixed frozen tissue or acidalcohol-fixed paraffin sections. 9,15 It is rarely possible to obtain specimens that meet with these ideal requirements, and if such standards were adhered to, valuable stored material would have to be excluded. Other factors such as quality of fixation and age of tissue also may influence immunohistochemical results. In this study, as in most other studies, immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissues. Our failure rate was two of six: immunohistochemistry failed to demonstrate epithelial antigens in two tumors that were subsequently shown to be of epithelial origin by electron microscopy. It is in these immunonegative tumors that ultrastructural studies are especially rewarding. In this report, the electron microscopic studies were valuable in showing that the tumor cells can induce reactive stromal proliferation where fibroblasts were seen in the conjunctival lesions and glial cells were present adjacent to the tumor cells in the involved retina. The admixture ofneoplastic and nonneoplastic stromal cells may render light microscopic diagnosis of such tumors difficult. We found both immunohistochemistry and electron microscopy to be valuable and complementary diagnostic techniques in this study of spindle cell carcinoma of the conjunctiva, a tumor that frequently poses a diagnostic problem because of its close morphologic similarity to sarcoma.
REFERENCES 1. Martin HE, Stewart FW. Spindle-cell epidermoid carcinoma. Am J Cancer 1935; 24:273-98. 2. Lichtiger B, Mackay B, Tessmer CF. Spindle-cell variant of squamous carcinoma: a light and electron microscopic study of 13 cases. Cancer 1970; 26:1311-20. 3. Feldman PS, Barr RJ. Ultrastructure of spindle cell squamous carcinoma. J Cutan Patho11976; 3:17-24. 4. Cohen BH, Green WR, Iliff NT, et al. Spindle cell carcinoma of the conjunctiva. Arch Ophthalmol 1980; 98: 1809-13. 5. Wise AC. A limbal spindle-cell carcinoma. Surv Ophthalmol1967; 12: 244-6. 6. BaUifora H. Spindle cell carcinoma: ultrastructural evidence of squamous origin and collagen production by the tumor cells. Cancer 1976; 37:2275-82. 7. Harris M. Differential diagnosis of spindle cell tumours by electron microscopy-personal experience and a review. Histopathology 1981; 5:81-105. 8. Woyke S, Domagala W, Olszewski W, Korabiec M. Pseudosarcoma of the skin: an electron microscopic study and comparison with the fine structure of the spindle-cell variant of squamous carcinoma. Cancer 1974; 33:970-80. 9. Messmer EP, Font RL. Applications of immunohistochemistry to ophthalmic pathology. Ophthalmology 1984; 91 :701-7. 10. GaUer KC, Alcock C, Heryet A, et al. The differential diagnosis of
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routinely processed anaplastic tumors using monoclonal antibodies. Am J Clin Pathol 1984; 82:33-43. Schlegel R, Banks-Schlegel S, McLeod JA, Pinkus GS. Immunoperoxidase localization of keratin in human neoplasms: a preliminary survey. Am J Patho11980; 101:41-50: Altmannsberger M, Osbom M, Schauer A, Weber K. Antibodies to different intermediate filament proteins: cell type-specific markers on paraffin-embedded human tissues. Lab Invest 1981; 45:427-34. Gabbiani G, Kapanci Y, Barazzone P, Franke WW. Immunohistochemical identification of intermediate-sized filaments in human neoplastic cells: a diagnostic aid for the surgical pathologist. Am J Pathol 1981; 104:206-16. Osbom M, Weber K. Intermediate filaments: cell-type-specific markers in differentiation and pathology. Cell 1982; 31 :303-6. Nagle RB, McDaniel KM, Clark VA, Payne CM. The use of antikeratin antibodies in the diagnosis of human neoplasms. Am J Clin Pathol 1983; 79:458-66. Cooper 0, Schermer A, Sun T-T. Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: Strategies, applications, and limitations. Lab Invest 1985; 52:243-56. Espinoza CG, Azar HA. Immunohistochemical localization of keratintype proteins in epithelial neoplasms: correlation with electron microscopic findings. Am J Clin Patho11982; 78:500-7. Heyderman E, Steele K, Ormerod MG. A new antigen on the epithelial membrane: its immunoperoxidase localisation in normal and neoplastic tissue. J Clin Patho11979; 32:35-9. Sloane JP, Ormerod MG. Distribution of epithelial membrane antigen in normal and neoplastic tissues and its value in diagnostic tumor pathology. Cancer 1981; 47:1786-95. Sloane JP, Hughes F, Ormerod MG. An assessment of the value of epithelial membrane antigen and other epithelial markers in solving diagnostic problems in tumour histopathology. Histochem J 1983; 15: 645-54. Zimmerman LE, Font RL, Tso MOM, Fine BS. Application of electron microscopy to histopathologic diagnosis. Trans Am Acad Ophthalmol Otolaryngol1972; 76:101-7. Sun T-T, Green H. Keratin filaments of cultured human epidermal cells: formation of intermolecular disulfide bonds during terminal differentiation. J Bioi Chem 1978; 253:2053-60. Chase DR, Weiss SW, Enzinger FM, Langloss JM. Keratin in epithelioid sarcoma: an immunohistochemical study. Am J Surg Patho11984; 8: 435-41.
24. Stemberger LA. Immunocytochemistry, 2nd ed. New York: John Wiley and Sons, 1979. 25. Hsu S-M, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981; 29:577-80. 26. Manglani KS, Manaligod JR, Ray B. Spindle cell carcinoma of the glans penis. a light and electron microscopic study. Cancer 1980; 46:2266-72. 27. Lazarides E.lntermediate filaments as mechanical integrators of cellular space. Nature 1980; 283:249-56. 28. Schlegel R, Banks-Schlegel S, Pinkus GS. Immunohistochemical localization of keratin in normal human tissues. Lab Invest 1980; 42: 91-6. 29. Osbom M. Components of the cellular cytoskeleton: A new generation of markers of histogenetic origin [Editorial]? J Invest Dermatol1984; 82:443-5. 30. Osbom M, Weber K. Tumor diagnosis by intermediate filament typing: a novel tool for surgical pathology. Lab Invest 1983; 48:372-94. 31. Thomas P, Said JW, Nash G, Banks-Schlegel S. Profiles of keratin proteins in basal and squamous cell carcinomas of the skin: an immunohistochemical study. Lab Invest 1984; 50:36-41. 32. Nathrath WBJ, Amholdt H, Wilson PD. Keratin, luminal epithelial antigen and carcinoembryonic antigen in human urinary bladder carcinomas. An immunohistochemical study. Pathol Res Pract 1982; 175:299307. 33. Delsol G, Stein H, Pulford KAF, et al. Human lymphoid cells express epithelial membrane antigen: implications for diagnosis of human neoplasms. Lancet 1984; 2: 1124-9. 34. Eichner R, Bonitz P, Sun T-T. Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression. J Cell Bioi 1984; 98:1388-96. 35. Holthiifer H, Miettinen A, Paasivuo R, et al. Cellular origin and differentiation of renal carcinomas: a fluorescence microscopic study with kidney-specific antibodies, antiintermediate filament antibodies, and lectins. Lab Invest 1983; 49:317-26. 36. Miettinen M, Franssila K, Lehto V-P, et al. Expression of intermediate filament proteins in thyroid gland and thyroid tumors. Lab Invest 1984; 50:262-70. 37. Ellis GL, Langloss JM, Enzinger FM. Coexpression of keratin and desmin in a carcinosarcoma involving the maxillary alveolar ridge. Oral Surg Oral Med Oral Patho11985; 60:410-6.
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Abstract: Six cases of conjunctival spindle cell carcinoma, a rare variant of squamous cell carcinoma, were studied. The median age of the three men and three women was 63.5 years. The tumors appeared as a single nodule in some patients or diffusely involved the conjunctiva in others. Two of the four individuals with intraocular extension presented with phthisis bulbi. Polyclonal anti keratin antibody was helpful and gave the most consistent results when compared with monoclonal anti keratin antibodies, AE1/3 and PKK1. The electron microscopic study of four lesions also established the epithelial nature of the tumor cells. Intracytoplasmic tonofilaments and a few desmosomes were present. Histopathologically, this variant of squamous cell carcinoma is difficult to distinguish from other spindle cell tumors, and this study demonstrates the value of immunohistochemistry and electron microscopy in supporting the correct diagnosis. Ophthalmology 1990; 97:711-717
Spindle cell carcinoma, a rare variant of squamous cell carcinoma, occurs in skin l - 3 and mucous membranes. 2,3 A review ofthe literature showed only three reported cases of spindle cell carcinoma involving the conjunctiva,4,5 and in one of these cases electron microscopy confirmed the diagnosis. Histopathologically, spindle cell carcinomas are difficult to distinguish from other spindle cell tumors, inOriginally received: October 2, 1989. Revision accepted: January 15, 1990. Department of Ophthalmology and Visual Sciences, Kentucky Lions Research Institute, University of Louisville, Louisville. 2 Drug Safety Division, Porer Central Research, Fort Washington. 3 Department of Ophthalmic Pathology, Armed Forces Institute of Pathol· ogy, Washington, DC. 1
Presented at the Association for Research in Vision and Ophthalmology Annual Meeting, Sarasota, May 1, 1985. The opinions and assertions contained herein are the private views of the authors and should not be construed as being official or representing the views of the Department of the Army or the Department of Defense. Reprint requests to Ahmed A. Hidayat, MD, Department of Ophthalmic Pathology, Armed Forces Institute of Pathology, Washington, DC 20306.
cluding amelanotic melanoma, malignant schwannoma, fibrosarcoma, leiomyosarcoma, and malignant fibrous histiocytoma. In the past, considerable reliance has been placed on the ultrastructural demonstration of epithelial differentiation to identify spindle cell carcinoma from other spindle cell tumors. 2- 6 Interpretation may be difficult,7 however, where the characteristic features are not always demonstrable ultrastructurally because of small samplings/,8 and the procedure is costly and time-consuming. More recently, immunohistochemistry has been used to aid in the classification of tumors in those cases in which morphologic features alone are insufficient for diagnosis. 9 ,lo We undertook this immunohistochemical study to determine the value of keratin and epithelial membrane antigen (EMA) as markers in identifying the spindle cell variant of squamous cell carcinoma of the conjunctiva. Recent studies have shown that the immunohistochemical detection of keratin 11-17 and EMA I8 - 20 offers a powerful tool for identifying tumors of epithelial origin. These markers are particularly valuable in recognizing spindle cell carcinoma and distinguishing it from other spindle cell tumors. Electron microscopy also is useful in establishing epithelial differentiation, and 711
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Table 1. Clinical Data for Six Patients with Spindle Cell Carcinoma of Conjunctiva Patient No. Sex/Age (yrs)
Clinical Appearance
Intraocular Extension
Treatment
1 F/30 2 Mj72 3 M/55 4 M/85 5 F/80 6 F/50
Unknown Limbal papillomas, phthisis bulbi Multiple conjunctival nodules, phthiSis bulbi Single limbal mass overlapping cornea Single nodule of bulbar conjunctiva Single nodule of bulbar conjunctiva
Yes Yes Yes No Yes No
Exenteration Enucleation Enucleation Enucleation Exenteration Excision
four of the six neoplasms in this study were studied ultrastructurally.
MATERIALS AND METHODS Twenty-four cases diagnosed as spindle cell tumor of the conjunctiva on file in the Registry of Ophthalmic Pathology at the Armed Forces Institute of Pathology were reviewed. Of these, only six had sufficient material (wet tissue, blocks, and/or unstained sections) for inclusion in this study. Hematoxylin-eosin-stained sections and special stains for melanin (either Fontana and/or Warthin-Starry at pH 3.3) were available for all cases. Five-micron glued sections offormalin-fixed, paraffin-embedded tissue were prepared for immunohistochemical studies. In four cases, 4% formaldehyde-fixed tissue was processed for electron microscopic studies. 21 Antibodies used for immunohistochemical studies were obtained in the following methods. Polyclonal antikeratin antiserum was derived in rabbits using keratin proteins isolated from human plantar calluses according to the method of Sun and Green. 22 The antibodies were affinity isolated using purified keratin proteins linked to cyanogen bromide-activated sepharose 4B (Pharmacia Chemical Co, Piscataway, NJ). The monoclonal keratin antibodies, PKKI (Labsystems-USA, Chicago, IL) and AEl/3 (Hybritech, San Diego, CA) were obtained from commercial sources. The monoclonal anti-EMA antibody also was obtained from a commercial source (Dako Laboratories, Copenhagen, Denmark; US Distributor, Accurate Chemical and Scientific Co, Westbury, NY). In an attempt to exclude other spindle cell tumors, two additional antibodies were used in this study: anti-S-lOO protein and antidesmin. Immunostaining for immunoreactive keratin, S-lOO protein, and desmin using polyclonal antibodies was performed as in previously described procedures. 23 The peroxidase antiperoxidase (PAP) method introduced by Sternberger4 was used. For the demonstration of keratin and desmin, sections were predigested with 0.1 % bovine trypsin (Sigma Chemical Co, St. Louis, MO) in 0.1M phosphate buffer, pH 7.8, at 37°C for 15 or 60 minutes. To reduce background staining, sections were incubated overnight in 5% normal swine serum. Sections were then incubated with appropriate primary antibodies at room temperature for 30 minutes, followed by 30-minute incubations with excess bridge antibody (swine anti-rabbit 712
Igs) and rabbit PAP complexes (Dako Laboratories, Copenhagen, Denmark) with intervening washes in O.OI-M phosphate-buffered saline, pH 7.4. Sections were developed in a solution containing 0.05% H 20 2 and 0.016% diaminobenzadine tetrahydrochloride (Sigma Chemical Co, St. Louis, MO) and counterstained with hematoxylin. Duplicate sections immunostained as above, except for the substitution of normal rabbit serum for the primary antibody, served as negative controls. For the monoclonal antibodies the avidin-biotin complex method (Vector Laboratories, Burlingame, CA) introduced by Hsu 25 was used. Predigested sections were incubated overnight in 5% horse serum and then incubated successively for 30 minutes at room temperature in monoclonal antibodies, biotinylated horse antimouse immunoglobulin (Vector Laboratories, Burlingame, CA), and avidin-biotin complex, with intervening washes in phosphate-buffered saline. Development of the chromogen and counterstaining was completed as described for the PAP method. Normal mouse serum was used as a negative control. In both the PAP and avidin-biotin complex techniques, uninvolved squamous epithelium served as an intrinsic positive control for the keratins and EMA.
RESULTS CLINICAL FINDINGS
The clinical data are summarized in Table 1. The median age ofthe six patients at the time of surgery was 63.5 years (range, 30-85 years). Three patients were male and three were female. The tumors originated in the conjunctiva as single or multiple nodules that were either located at the limbus or diffusely involved the conjunctiva more extensively. One tumor in our series (case 4) was a large sessile limbal mass that overlapped the cornea and obscured vision (Fig 1), and two others (cases 5 and 6) had single nodules of the bulbar conjunctiva. In the other two patients, the conjunctiva was more diffusely involved, and in case 3 the entire conjunctival sac was studded with small tumor nodules. The corneoscleral area was invaded to a varying extent in all cases. In four patients, there was intraocular extension, and in two of these (cases 2 and 3) the tumors were predominantly intraocular and the clinical diagnosis was phthisis bulbi. Both of the patients with phthisis bulbi had protracted and complicated ocular histories, which diverted attention from the conjunctival
Fig 1. Top left, a large and fleshy conjunctival mass in the right eye overlaps a scarred and vascularized cornea, Courtesy of Professor J. S. Jain (AFIP Neg. 86-5207). Fig 2. Second row left, diffuse conjunctival tumor that has invaded the limbus. Perforation of a corneal ulcer is present (hematoxylin-eosin; original magnification, X3.5, AFIP Neg. 86-5231). Fig 3. Top right, large intraocular mass involves the vitreous and detached retina (hematoxylineosin; original magnification, X3.5, AFIP Neg. 86-5221). Fig 4. Third row left, limbal epithelium shows acanthosis and loss of polarity of the abnormal cells that extend to the surface. These pathologic changes are consistent with carcinoma in situ (hematoxylin-eosin; original magnification, X300, AFIP Neg. 865219). Fig 5. Third row right, hypercellular tumor is composed of spindle-shaped cells, arranged in interlacing fascicles. The tumor resembles fibrosarcoma or fibrous histiocytoma (hematoxylineosin; original magnification, X75, AFIP Neg. 86-5229). Fig 6. Fourth row left, atypical spindle-shaped cells have elongated and vesicular nuclei with irregular nuclear membrane and prominent nucleoli. Abnormal mitotic figures are present (hematoxylin-eosin; original magnification, X630, AFIP Neg. 865233). Fig 7. Bottom right, some of the pleomorphic and bizarre tumor cells have hyperchromatic nuclei and dense eosinophilic cytoplasm. The stroma is infiltrated by inflammatory cells (hematoxylin-eosin; original magnification, X630, AFIP Neg. 86-5220). Fig 8. Bottom left, spindle-shaped tumor cells show positive staining for polyclonal antikeratin. The loose and edematous stroma is infiltrated by inflammatory cells (peroxidase-antiperoxidase technique; original magnification, X450, AFIP Neg. 86-5223).
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Table 2. Immunohistochemical Results of Anti-epithelial Antibodies Cases
Polyclonal anti keratin AE1/3 PKK1 Anti-EMA
+ +
2
3
4
+ + +
+
+
5
6
+ +
pathology. In case 2, the patient had a 9-year history of recurrent herpetic keratitis for which multiple intraocular surgical procedures had been undertaken. Conjunctival papillomas had been noted 3 years before enucleation but a biopsy had been done. It is likely that the surgical procedures, which included three penetrating keratoplasties, cataract extraction, and vitrectomy, contributed to the intraocular spread of the tumor. The other patient with a phthisical eye (case 3) had a history of unspecified ocular inflammation 40 years before enucleation. The entire conjunctival sac in this case was extensively thickened and distorted by many rounded nodules. All patients had been treated surgically either by local excision ofthe tumor, enucleation, or exenteration. Follow-up data were incomplete because some patients were new and others were lost to follow-up.
Fig 9. A desmosome (arrow) between tumor cells is present. The nuclear membrane is irregular (original magnification, X35,000, AFIP Neg. 869160).
LIGHT MICROSCOPIC FINDINGS
The tumors appeared as a single polypoid limbal mass or with diffuse involvement of the entire bulbar conjunctiva (Fig 2). In one lesion, both the bulbar and tarsal conjunctivae were invaded by the tumor in the form of multiple small nodules. In one phthisical eye that had undergone multiple surgical procedures, including keratoplasty, before enucleation the large intraocular tumor involved the vitreous and detached gliotic retina (Fig 3). In that eye, the limbal epithelium showed abnormal changes consistent with carcinoma in situ (Fig 4), and serial sections showed invasion of the transplanted corneal stroma by atypical spindle-shaped cells. The tumors were composed of spindle-shaped cells arranged haphazardly in interlacing fascicles (Fig 5) or were widely separated by edematous-appearing stroma containing many chronic inflammatory cells. The nuclei of the tumor cells were mostly elongated and vesicular with an irregular nuclear membrane and prominent nucleoli (Figs 6, 7). Few pleomorphic and bizarre cells with hyperchromatic nuclei and dense eosinophilic cytoplasm also were present (Fig 7). Mitotic figures were frequent (Figs 6, 7). Continuity between the tumor and the overlying epithelium was noted in four lesions. In the remaining two lesions, there was surface ulceration. Special stains for melanin (Warthin-Starry and Fontana) were negative in all tumors. IMMUNOHISTOCHEMICAL STUDIES
The immunohistochemical results are summarized in Table 2. Immunohistochemical staining for epithelial an714
tigens was successful in four of the six tumors (cases 14). There was some variation in the staining intensity, but positive cells were readily recognized by the characteristic gold-brown to red-brown staining product. The polyclonal antikeratin antibody decorated cells in four lesions (Fig 8). Identifiable keratin was focal in three of these tumors and diffuse in one. Of the monoclonal antibodies, PKK 1decorated cells in two tumors, anti-EMA in two, and AE1/ 3 in one. Immunostaining with the monoclonal antibodies resulted in focal, less-intense staining than with the polyclonal antikeratin antibody. Uninvolved conjunctival epithelium served as an intrinsic control for epithelial antigens and was positive in all six cases. The immunologic markers S-1 00 protein and desmin were not demonstrated in any of the lesions studied. ELECTRON MICROSCOPIC STUDIES
Tumor cells were predominantly spindle shaped with elongated nuclei and irregular nuclear membranes. A few desmosomes were present (Fig 9) between adjacent cells, but in many areas where the cells were separated from each other by edematous stroma the desmosomes were absent. The cytoplasm contained rough-surfaced endoplasmic reticulum, few mitochondria, and variable amounts of to no filaments (Fig 10). In addition to the tumor cells, there was evidence of reactive stromal proliferation where fibroblasts were seen in the conjunctival lesions and glial cells were present adjacent to the tumor cells in the involved retina.
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Fig 10. The cytoplasm contains many bundles of tonofilaments (T) and rough-surfaced endoplasmic reticulum (original magnification. X21,OOO, AFIP Neg. 9162).
DISCUSSION In this report, the median age of the patients at the time of surgery was 63.5 years and the sex incidence was equal. The tumor usually appears clinically as single or multiple nodules located at the limbus, as in previous reports,4,5 but in some patients it may involve the conjunctiva more diffusely. It is interesting that two of the patients in our study (cases 2 and 3) had conjunctival papillomas and later developed phthisis bulbi. A primary conjunctival tumor with intraocular extension was an unsuspected finding. Histopathologically, the interpretation of the intraocular findings in these two cases was extremely difficult. In one lesion (case 2), the main part of a cellular mass was intimately related to a detached and gliotic retina, and the differential diagnosis was between massive retinal gliosis and adenocarcinoma of the retinal pigment epithelium. In the other case of phthisis bulbi (case 3), adenocarcinoma of the nonpigmented ciliary epithelium was the initial diagnosis. The differential diagnoses in the other four patients included amelanotic melanoma, Kaposi's sarcoma, leiomyosarcoma, and malignant schwan noma, in addition to spindle cell carcinoma. Although conclusions on tumor behavior cannot be drawn from this study because of the lack of follow-up data, the fact that three patients required enucleation and two required exenteration is indicative of local aggressiveness. This is in agreement with other studies,4,5 and a
wide excision of the initial lesion is recommended to avoid recurrences. Before the application of immunohistochemical techniques to tumor diagnosis, the diagnosis of spindle cell carcinoma rested on characteristic histologic and ultrastructural features. The most consistent criterion for histologic diagnosis is continuity of the spindle cell tumor with the surface epithelium, although a careful search through numerous sections is often required to demonstrate this feature. Cellular atypia and even frank malignant change within the overlying epithelium also may be present. The observation of both these changes is dependent on an intact surface epithelium. Unfortunately, surface ulceration with loss of these valuable clues is a common feature of this neoplasm. In this study, the conjunctiva in case 2 showed changes consistent with carcinoma in situ. Transition from spindle to squamous cells may be seen, but this is an inconsistent feature and was not observed in any of our cases. The diagnosis of spindle cell carcinoma may be reliably made by the ultrastructural presence of to no filaments and desmosomes, although extensive sampling is often needed to demonstrate these structures. 6 ,26 The recent literature contains several reports that indicate that the cytoplasmic keratins ll - 17 and epithelial membrane antigen l8 - 2o are reliable diagnostic markers for tumors of epithelial origin. The keratins, 16,27.28 which are present in almost all epithelial cells, are a group of at least 19 closely related polypeptides. They form one of the five 715
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classes of cytoskeletal intermediate filament proteins that are immunologically and biochemically distinct. 27,29 Many investigators have reported that the filaments of neoplastic cells mirror those of the cell of tumor origin,13,14,30 and the development of specific antisera to intermediate filament proteins has become a useful adjunct to distinguish morphologically similar tumors, Keratins have been demonstrated in a number of tumors that affect the ocular adnexa, including basal cell,1l,13,15,17,31 squamous cell 11,13,15,17,31 sebaceous cell 17 and mucoepidermoid carcin~mas, 17 Epithelial membrane antigen, or milk fat globule membrane antigen, is a large molecular weight glycoconjugate that also is widely distributed in normal epithelia and tumors derived from them. 12,18,20,32 Although EMA is not always demonstrable in normal squamous epithelium, alteration by either nonneoplastic or neoplastic disease processes may result in its expression. 19 There is recent evidence that expression of EMA is not restricted to epithelial cells but also may be demonstrated on reactive and neoplastic plasma cells and some lymphomas. 33 In this study, the polyclonal antikeratin antibody was helpful and gave the most consistent results with positive staining of cells in four tumors, whereas the monoclonal antikeratin antibodies, AEl/3 and PKKl, were positive in only one and two tumors respectively. The polyclonal antiepidermal keratin antibody is the antikeratin that has been used most frequently in similar studies. The monoclonal antikeratin antibodies are a more recent introduction. The antikeratin AEl/3 is composed of monoclonal antibodies AEI and AE3, which recognize most acidic and basic keratin proteins. 16,34 The other monoclonal antikeratin we used was PKK1, a monoclonal antibody raised against a pig kidney epithelial cell line, which is directed against low molecular weight keratin proteins found principally in simple epithelia. 35 ,36 In a study on renal carcinoma, PKK 1, used in conjunction with a polyclonal antikeratin antibody, provided valuable information on the cellular origin and differentiation of these tumors.35 In another study, PKKI was found to be less helpful than the polyclonal antikeratin antibody and the monoclonal mixture, AEI/3. 37 We found only two of the six tumors stained with the monoclonal anti-EMA antibody. Although it has been reported that squamous epithelium does not express EMA,19 we did not find this to be true because EMA was demonstrated in the overlying squamous epithelium in all six tumors included in this study. Immunohistochemistry has a number of advantages over electron microscopy. A larger area of tumor can be examined, thus avoiding the introduction of error because of inadequate sampling in ultrastructural studies. In electron microscopy, several blocks of tumor tissue often have to be examined to find tonofilaments and desmosomes, the characteristic ultrastructural features of epithelial cells, because stromal cells may be indistinguishable from tumor cells in all other respects. Tonofilaments are the ultrastructural equivalent of keratin, and the focal expression of keratin, demonstrated immunohistochemically in the keratin-positive tumors, correlates with the sparsity oftu716
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mor cells with ultrastructural evidence of squamous differentiation reported by various authors. 7,26 Immunohistochemistry also is less time-consuming than electron microscopy. One disadvantage of immunohistochemistry is that the fixation method influences the sensitivity of the technique. Optimal results with immunohistochemistry are achieved by using either unfixed frozen tissue or acidalcohol-fixed paraffin sections. 9,15 It is rarely possible to obtain specimens that meet with these ideal requirements, and if such standards were adhered to, valuable stored material would have to be excluded. Other factors such as quality of fixation and age of tissue also may influence immunohistochemical results. In this study, as in most other studies, immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissues. Our failure rate was two of six: immunohistochemistry failed to demonstrate epithelial antigens in two tumors that were subsequently shown to be of epithelial origin by electron microscopy. It is in these immunonegative tumors that ultrastructural studies are especially rewarding. In this report, the electron microscopic studies were valuable in showing that the tumor cells can induce reactive stromal proliferation where fibroblasts were seen in the conjunctival lesions and glial cells were present adjacent to the tumor cells in the involved retina. The admixture ofneoplastic and nonneoplastic stromal cells may render light microscopic diagnosis of such tumors difficult. We found both immunohistochemistry and electron microscopy to be valuable and complementary diagnostic techniques in this study of spindle cell carcinoma of the conjunctiva, a tumor that frequently poses a diagnostic problem because of its close morphologic similarity to sarcoma.
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